turner/weber/shen laboratory departments of pathology and ... vectors after nov 1... · friday,...

Turner/Weber/Shen Laboratory Departments of Pathology and Surgery The University of Chicago turnerlab.bsd.uchicago.edu © April 5, 2013

Plasmid #855

Plasmid name pPBH-P-TREtight-EGFP-C

Plasmid description:

Name pPBH-P-TREtight-EGFP-C

Brief Description of the plasmid:

This is a PiggyBac vector for tet-inducible eukaryotic protein expression of N-terminal tagged EGFP fusion proteins. Between the inverted terminal repeat sequences, the plasmid contains the hygromycin resistant gene expression cassette for eukaryotic selection. 3’ to the hygromycin resistant cassette, it contains a human phosphoglycerate kinase 1 (PGK) promoter driven TetOn3G expression cassette. 5’ to the hygromycin resistant cassette, it contains a TREtight promoter driven EGFP expression cassette. Immediately 3’ to the EGFP, it contains a multiple cloning site to allow insertion of protein coding DNA sequences.

When transfected into eukaryotic cell lines, the TetON3G transactivator is constitutively expressed. In the presence of doxycycline, the TetON3G transactivator binds to the TREtight promoter within the plasmid to drive EGFP or EGFP-fusion protein expression in a dose dependent fashion. Such inducible expression can be achieved in both transiently or stably transfected cells.

The only difference between this vector and plasmid #734, pPBH-TREtight-EGFP-C is the usage of PGK promoter instead of EF-1� promoter to drive TetON3G protein expression. Because the PGK promoter is generally much weaker than EF-1� promoter, this leads to relatively low TetON3G expression in transfected cells. Use of this vector over #734 may limit cellular changes induced by TetON3G. Although this may also lead to reduced Tet-induced expression, PGK promoter activity is sufficient to support high levels of Tet-inducible protein expression in Caco-2 cells.

When co-transfected with pSPB-transposase (plasmid #709), the PiggyBac transposase expressed from pSPB-transposase will recognize the inverted terminal repeat and insert the DNA sequence between these terminal repeats into TTAA sites of the host cell chromosomes, allowing site-specific integration of plasmid DNA with high efficiency.

This vector has a spectinomycin resistant gene expression cassette for bacterial amplification.

Summary

1. Cloning Strategy

The 1.4 kb TRE-Tight-EGFP gene expression cassette was PCR amplified from the pPBH-TREtight-EGFP-C (plasmid #734). Purified PCR product was inserted into the SfiI site of pPBH-PGK-TetON3G (plasmid #851) by using a directional ligation independent cloning kit (InFusion HD cloning kit, Clontech 639645). Successful insertion was confirmed by restriction digestion with and sequencing.

2. In silico compilation of the plasmid sequence:

The plasmid sequence listed in this document was generated by combining the sequencing results and the preexisting sequence of pPBH-PGK-TetON3G (plasmid #851). The plasmid was not completely sequenced.

This plasmid contains the following unique restriction enzyme recognizing sites within the multiple cloning site for insertion of protein coding DNA sequences. Acc65I, KpnI, NheI, BmtI, NotI, ClaI Commonly used restriction enzyme recognizing sequences are highlighted with in the sequence of the multiple cloning site: KpnI, NheI, NotI, ClaI TCCGGACTCAGATCTCGAGCTCAAGCTTCGAACTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCTCTAGTCAGCTGACGCGTGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTCTAGA Key Features:

5’ core insulator 264..502 3’ PiggyBac terminal repeat 25..257 P tight tet-responsive promoter 524..839 EGFP-MCS 864..1708 Multiple cloning site (MCS) 1581..1708 HSV TK promoter 2297..3334 hygromycin resistant gene 2297..3334 HSV TK polyadenylation sequence 2297..3334 PGK promoter 3879..4516 Tet-On 3G 4517..5263 SV40 polyadenylation sequence 5286..5728 3’ core insulator 5993..6260 5’ PiggyBac terminal repeat 6261..6295 Spectinomycin resistant gene 8638..9666

Primers:

x pMPB F81-104 5’-ACGCATGTGTTTTATCGGTCTGTAT-3’ binds to 81..105 of pPBH-TREtight-EGFP-C

x EGFP beginning R 5’-GCCGGTGGTGCAGATGAACT-3’ binds to complement(1000..1019)of pPBH-TREtight-EGFP-C

x EGFP 1/4F 5’-TTCAAGGACGACGGCAACTACAAG-3’ binds to 1164..1187 of pPBH-TREtight-EGFP-C

x EGFP-C sequencing primer (Clontech #6478-1)

5’-CATGGTCCTGCTGGAGTTCGTG-3’ binds to 1517..1538 of pPBH-TREtight-EGFP-C This primer can be used to sequence DNA sequences inserted into the multiple cloning site.

x TK prom 1/3 R 5’-TGGGCGGGGTTTGTGTCATCATAGA-3’

binds to 2112..2136 of pPBH-TREtight-EGFP-C

x hygro 1/3R 5’-ACAGCGGGCAGTTCGGTTTCAG-3’ binds to 2607..2628of pPBH-TREtight-EGFP-C

x hygro 1/3F 5’-CGCAAGGAATCGGTCAATACACT-3’ binds to 2709..2731of pPBH-TREtight-EGFP-C

x hygro 2/3F 5’-CCGTGGTTGGCTTGTATGGA-3’ binds to 3005..3024of pPBH-TREtight-EGFP-C

x TKpolyA 1/2F 5’ATGGTTTATGGTTCGTGGGGGTTATTCTTT-3’ binds to 3631..3660of pPBH-TREtight-EGFP-C

x TET-ON 3G 1/2F 5’-CACTGGGCTGCGTATTGGAGGAACA-3’ binds to 4938..4962 of pPBH-TREtight-EGFP-C

x SV40polyA 1/2F 5’-GGCCGGACCACGCTATCTGT-3’ binds to 5533..5552of pPBH-TREtight-EGFP-C

x pMPB R 2549-2523 5’-CGCATGATTATCTTTTACGTGACTTTT-3’ binds to 6077..6103of pPBH-TREtight-EGFP-C

x pMPB F 2563-2582 5’-CGCGGTCGTTATAGTTCAAA-3’ binds to 6117..6136of pPBH-TREtight-EGFP-C

x pMPB F 6058-6083 5’-GAGATCACCAAGGTAGTCGGCAAATA-3’ binds to 9622..9647of pPBH-TREtight-EGFP-C

Diagnostic Digests

AflII and KpnI (double digest) 305bp and 9384 bp

HindIII: 9689bp

Plasmid History

Plasmid generated by Le Shen

Date plasmid generated 12/2012

Data file generated by Amulya Lingaraju and Le Shen

Data file generated 01/2013,

Data file version history V1.0, 01/2013

Plasmid map:

Plasmid sequence:

CTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGAGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCACGAGGCCCTTTCGTCTTCACTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTATCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCT

Cor...ns

er

EGFP

S...A

hyg

rom

yci

nre

sis

tan

tg

en

e

HS

...er

NheIA

hdI

NsiI

PfoI

PciI

Sa

pI

Zra

I

AflII

Asi

SI

S

KpnI

Bsp

HI

MscI

ClaI

Bm

tI

Acc65I

SanDI

AclI

BstXI

SspI

PshA

IA

at II

NotI

1000

2000

3000

6000

70

00

8000

HSVFr..

.sig

nal

BsrG

I

GGAGAATTCTACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCGAACTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCTCTAGTCAGCTGACGCGTGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTCTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCCTCGAGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCTTAAGCGGCCAGACTAGTTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCCTGCTTCATCCCCGTGGCCCGTTGCTCGCGTTTGCTGGCGGTGTCCCCGGAAGAAATATATTTGCATGTCTTTAGTTCTATGATGACACAAACCCCGCCCAGCGTCTTGTCATTGGCGAATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCGAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCGCTTAACAGCGTCAACAGCGTGCCGCAGATCAGCTTGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGGATCGGGAGATGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCCACTGGCCCCGTGGGTTAGGGACGGGGTCCCCCATGGGGAATGGTTTATGGTTCGTGGGGGTTATTCTTTTGGGCGTTGCGTGGGGTCAGGTCCACGACTGGACTGAGCAGACAGACCCATGGTTTTTGGATGGCCTGGGCATGGACCGCATGTACTGGCGCGACACGAACACCGGGCGTCTGTGGCTGCCAAACACCCCCGACCCCCAAAAACCACCGCGCGGATTTCTGGCGTGCCAAGCTCCGACCCCCAAAAACCACCGCGCGGATTTCTGGCGTGCCAAGCTTGGTGCGTACAATTAAGGGATTATGGTAAATCCACTTACTGTCTGCCCTCGTAGCCATCGAGATAAACCGCAGTACTCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCCAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAACTAAGTAAGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCTGCAAGCCTCGTCGTCCTGGCCGGACCACGCTATCTGTGCAAGGTCCCCGGCCCCGGACGCGCGCTCCATGAGCAGAGCGCCCGCC

GCCGAGGCGAAGACTCGGGCGGCGCCCTGCCCGTCCCACCAGGTCAACAGGCGGTAACCGGCCTCTTCATCGGGAATGCGCGCGACCTTCAGCATCGCCGGCATGTCCCCCTGGCGGACGGGAAGTATCCAGCTCGACCAACCATTGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTGTACCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTACCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTGGACATAAGCCTGTTCGGTTCGTAAGCTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATTATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAACCCTCGAGCCACCCATGACCAAAATCCCTTAACGTGAGTTA

Friday, April 5, 2013 12:14 AM Page 1 of 15p855data

BsiWI

CTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATG5'

3' PB-TR

70

CGTAAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTAA5'

3' PB-TR

xpMPB F81-104

140

GTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTA5'

3' PB-TR

210

TTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGAGGGACAGCCCCCCCC5'

Core Ins

3' PB-TR

280

CAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGC5'

Core Ins

350

TCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTG5'

Core Ins

420

GCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGG5'

Core Ins

490

GAAAAGGCCTCACGAGGCCCTTTCGTCTTCACTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTC5'

Core Ins P tight tet-responsive promoter

TetO 19mer

560


GAGTTTACTCCCTATCAGTGATAGAGAACGATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGT5'

P tight tet-responsive promoter

TetO 19 mer

TetO 19mer

630

CGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTAT5'


TetO 19mer

TetO 19mer

700

GTCGAGTTTATCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTA5'


TetO 19mer

TetO 19 mer

770

TGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCT5'

pCEP_fwd_primer

LNCX_primer


P min CMV delta

TATA box

840

GGAGAATTCTACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCT5'

EGFP

910

GGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACC5'

EGFP

980

TACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGA5'

EGFP

x EGFP beginning R

1050

CCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAA5'

EGFP

1120


PfoI

GTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACC5'

EGFP

x EGFP 1/4F

1190

CGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGG5'

EGFP

1260

AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGA5'

EGFP

1330

CAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTC5'

EGFP

1400

GCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGA5'

EGFP

1470

GCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGAC5'

EGFP

EGFP-C primer

1540

BsrGI

CGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCG5'

EGFP

multiple cloning site

1610

NotIBmtI

NheIKpnIAcc65I

AACTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCTCTAGTCAGCTGACGCGTGCTAGCGCGGCCGCAT5'

EGFP


1680


ClaI

CGATAAGCTTGTCGACGATATCTCTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTG5'

EGFP

SV40 polyA


1750

CTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTT5'

SV40 polyA

1820

GTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTT5'

SV40 polyA

1890

AflII

TCACTGCCTCGAGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCTTAAGCGGCCAGACTAGTTTGGTTT5'

SV40 polyA

1960

GCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTG5'

2030

CCGCCCTGCTTCATCCCCGTGGCCCGTTGCTCGCGTTTGCTGGCGGTGTCCCCGGAAGAAATATATTTGC5'

HSV TK promoter

2100

ATGTCTTTAGTTCTATGATGACACAAACCCCGCCCAGCGTCTTGTCATTGGCGAATTCGAACACGCAGAT5'

HSV TK promoter

xTK prom 1/3 R

2170

GCAGTCGGGGCGGCGCGGTCCGAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACACCGAG5'

HSV TK promoter

2240

CGACCCTGCAGCGACCCGCTTAACAGCGTCAACAGCGTGCCGCAGATCAGCTTGATATGAAAAAGCCTGA5'

hygromycin...tant geneHSV TK promoter

M K K P E

1 2 3 4 5

2310


AatIIPshAI

ZraI

ACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTC5'

hygromycin resistant gene

L T A T S V E K F L I E K F D S V S D L M Q L

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

2380

TCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATA5'


S E G E E S R A F S F D V G G R G Y V L R V N

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

2450

GCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCC5'


S C A D G F Y K D R Y V Y R H F A S A A L P I P

52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

2520

GGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTC5'


E V L D I G E F S E S L T Y C I S R R A Q G V

76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98

2590

ACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGA5'


xhygro 1/3 R

T L Q D L P E T E L P A V L Q P V A E A M D A

99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121

2660

AsiSI

TCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACAC5'


xhygro 1/3F

I A A A D L S Q T S G F G P F G P Q G I G Q Y T

122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145

2730


TACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGAC5'


T W R D F I C A I A D P H V Y H W Q T V M D D

146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168

2800

ACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCC5'


T V S A S V A Q A L D E L M L W A E D C P E V

169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191

2870

GGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCAT5'


R H L V H A D F G S N N V L T D N G R I T A V I

192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215

2940

TGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGG5'


xhy.../3F

D W S E A M F G D S Q Y E V A N I F F W R P W

216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238

3010

TTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGC5'


xhygro 2/3F

L A C M E Q Q T R Y F E R R H P E L A G S P R

239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261

3080

TCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGA5'


L R A Y M L R I G L D Q L Y Q S L V D G N F D D

262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285

3150

TGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAA5'


A A W A Q G R C D A I V R S G A G T V G R T Q

286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308

3220


ATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGAC5'


I A R R S A A V W T D G C V E V L A D S G N R

309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331

3290

GCCCCAGCACTCGTCCGGATCGGGAGATGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAG5'


HSV Fragment containing TK poly A signal

R P S T R P D R E M G E A N .

332 333 334 335 336 337 338 339 340 341 342 343 344 345 346

3360

GAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAAC5'


3430

GCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCG5'


3500

CGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGG5'


3570

SanDI

GCGGCAGGCCCTGCCATAGCCACTGGCCCCGTGGGTTAGGGACGGGGTCCCCCATGGGGAATGGTTTATG5'


xTKpolyA 1/2F

3640

GTTCGTGGGGGTTATTCTTTTGGGCGTTGCGTGGGGTCAGGTCCACGACTGGACTGAGCAGACAGACCCA5'


xTKpolyA 1/2F

3710

TGGTTTTTGGATGGCCTGGGCATGGACCGCATGTACTGGCGCGACACGAACACCGGGCGTCTGTGGCTGC5'


3780

CAAACACCCCCGACCCCCAAAAACCACCGCGCGGATTTCTGGCGTGCCAAGCTCCGACCCCCAAAAACCA5'


3850


CCGCGCGGATTTCTGGCGTGCCAAGCTTGGTGCGTACAATTAAGGGATTATGGTAAATCCACTTACTGTC5'

human PGK promoter

3920

TGCCCTCGTAGCCATCGAGATAAACCGCAGTACTCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATC5'

human PGK promoter

3990

TACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTT5'

human PGK promoter

4060

GGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTT5'

human PGK promoter

4130

CTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTC5'

human PGK promoter

4200

GCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTG5'

human PGK promoter

4270

AGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCA5'

human PGK promoter

4340

GAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGC5'

human PGK promoter

4410

ACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCC5'

human PGK promoter

4480

CAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACT5'

human PGK promoter Tet-On 3G

M S R L D K S K V I N

1 2 3 4 5 6 7 8 9 10 11

4550

CTGCTCTGGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGG5'

Tet-On 3G

S A L E L L N G V G I E G L T T R K L A Q K L G

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

4620


PmlI

AGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGCCAATCGAG5'

Tet-On 3G

V E Q P T L Y W H V K N K R A L L D A L P I E

36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

4690

ATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACA5'

Tet-On 3G

M L D R H H T H S C P L E G E S W Q D F L R N

59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81

4760

NruI

ACGCCAAGTCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCC5'

Tet-On 3G

N A K S Y R C A L L S H R D G A K V H L G T R P

82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

4830

PsrI

AACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAG5'

Tet-On 3G

T E K Q Y E T L E N Q L A F L C Q Q G F S L E

106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128

4900

PsrI'

AACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATC5'

Tet-On 3G

xTET-ON 3G 1/2F

N A L Y A L S A V G H F T L G C V L E E Q E H

129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151

4970

AAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAAACAAGCAATTGA5'

Tet-On 3G

Q V A K E E R E T P T T D S M P P L L K Q A I E

152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175

5040


AloI'AloI

GCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAG5'

Tet-On 3G

L F D R Q G A E P A F L F G L E L I I C G L E

176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198

5110

AAACAGCTAAAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAG5'

Tet-On 3G

K Q L K C E S G G P T D A L D D F D L D M L P

199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221

5180

CCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACAT5'

Tet-On 3G

A D A L D D F D L D M L P A D A L D D F D L D M

222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245

5250

GCTCCCCGGGTAACTAAGTAAGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACT5'

Tet-On 3G SV40 polyA

L P G .

246 247 248 249

5320

AGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAA5'

SV40 polyA

5390

GCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGA5'

SV40 polyA

5460

GGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCTGCAAGCCTCGTCGTC5'

SV40 polyA

5530

CTGGCCGGACCACGCTATCTGTGCAAGGTCCCCGGCCCCGGACGCGCGCTCCATGAGCAGAGCGCCCGCC5'

SV40 polyA

xSV40polyA 1/2F

5600


SexAI

GCCGAGGCGAAGACTCGGGCGGCGCCCTGCCCGTCCCACCAGGTCAACAGGCGGTAACCGGCCTCTTCAT5'

SV40 polyA

5670

CGGGAATGCGCGCGACCTTCAGCATCGCCGGCATGTCCCCCTGGCGGACGGGAAGTATCCAGCTCGACCA5'

SV40 polyA

5740

BstXI

ACCATTGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTC5'

5810

AGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGC5'

5880

GGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAA5'

5950

TTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATATATAATAAG5'

Core Ins

6020

TTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAA5'

Core Ins

xpMPB R 2549-2523

6090

AGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGAC5'

Core Ins

xpMPB R 2549-2523

xpMPB F 2563-2582

6160

AAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTAT5'

Core Ins

6230

NsiISspI

TTAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAAT5'

Core Ins 5' PB-TR

6300

CTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGCTATCTG5'

6370


GGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGG5'

6440

MscI

ATGACTGCTGCTGCATTGACGTTGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCAT5'

6510

GCACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACA5'

6580

CAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGGAACTGCCGTG5'

6650

CCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGGGTATCCTGGCTG5'

6720

GATGCCGCAGAAATGGACATGGATACCCCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCAT5'

6790

AGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTG5'

6860

TAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAG5'

6930

TCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTG5'

7000

SapI

GGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC5'

7070

PciI

TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAA5'

7140

GGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTG5'

7210

ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC5'

7280

GTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC5'

7350

TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG5'

7420

TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA5'

7490


TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGC5'

7560

AGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGA5'

7630

CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGG5'

7700

CAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGA5'

7770

TCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGA5'

7840

BspHI

TTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC5'

7910

AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA5'

7980

AhdI

GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG5'

8050

GCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGC5'

8120

AATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT5'

8190

AclI

ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTG5'

8260

CTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG5'

8330

GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA5'

8400

AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCAT5'

8470

CCGTAAGATGCTTTTCTGTGACTGGTGAGTGTACCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTACC5'

8540

CAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTGGACATAAGCCTGTTCG5'

8610


GTTCGTAAGCTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCG5'

SpnR

8680

GTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCG5'

SpnR

8750

GGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACG5'

SpnR

8820

CAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATTATGAGGGAAGCGGTGATCGCCGAAGTATCGACTC5'

SpnR

8890

AACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGG5'

SpnR

8960

CTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTT5'

SpnR

9030

BclI

GATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGA5'

SpnR

9100

TTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCG5'

SpnR

9170

CGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGAC5'

SpnR

9240

ATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGG5'

SpnR

9310

AACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTC5'

SpnR

9380


GCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTA5'

SpnR

9450

ACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGC5'

SpnR

9520

CCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCA5'

SpnR

9590

GTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAACCCTCGAGCCAC5'

SpnR

xpMPB F 6058-6083

9660

CCATGACCAAAATCCCTTAACGTGAGTTA5'

SpnR

9689

turner/weber/shen laboratory departments of pathology and ... vectors after nov 1... · friday,...

Documents