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Ef1a_Large T-antigen_Ires_Puro vector (V006660#)

Basic Vector Information

Basic Vector Information
Vector Name Ef1a_Large T-antigen_Ires_Puro Antibiotic Resistance Ampicillin
Length 11672 bp Type Mammalian Expression, Lentiviral, Cre/Lox
Selection Marker Puromycin Cloning Method Restriction Enzyme
5' Primer Ef1a

Ef1a_Large T-antigen_Ires_Puro vector Vector Map

Ef1a_Large T-antigen_Ires_Puro11672 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000105001100011500large T antigenIRES2PuroRloxP5' LTR (truncated)BGH-revf1 oripBABE 3'EM7 promoterBleoRSV40 poly(A) signalM13 revlac operatorlac promoterCAP binding siteL4440oriAmpRAmpR promoterpRS-markerCMV enhancerCMV promoter5' LTR (truncated)HIV-1 PsiRREgp41 peptidecPPT/CTSEF-1-alpha promoterT7

Plasmid Resuspension protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

Ef1a_Large T-antigen_Ires_Puro vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported               11672 bp ds-DNA     circular SYN 16-AUG-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Ef1a_Large T-antigen_Ires_Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11672)
  AUTHORS   Mali P, Ye Z, Hommond HH, Yu X, Lin J, Chen G, Zou J, Cheng L
  TITLE     Improved efficiency and pace of generating induced pluripotent stem 
            cells from human adult and fetal fibroblasts.
  JOURNAL   Stem Cells. 2008 Aug . 26(8):1998-2005.
  PUBMED    18511599
REFERENCE   2  (bases 1 to 11672)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Stem Cells.
            2008 Aug . 26(8):1998-2005."
FEATURES             Location/Qualifiers
     source          1..11672
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..2127
                     /codon_start=1
                     /product="large T antigen from SV40 (simian virus 40)"
                     /label=large T antigen
                     /translation="MDKVLNREESLQLMDLLGLERSAWGNIPLMRKAYLKKCKEFHPDK
                     GGDEEKMKKMNTLYKKMEDGVKYAHQPDFGGFWDATEIPTYGTDEWEQWWNAFNEENLF
                     CSEEMPSSDDEATADSQHSTPPKKKRKVEDPKDFPSELLSFLSHAVFSNRTLACFAIYT
                     TKEKAALLYKKIMEKYSVTFISRHNSYNHNILFFLTPHRHRVSAINNYAQKLCTFSFLI
                     CKGVNKEYLMYSALTRDPFSVIEESLPGGLKEHDFNPEEAEETKQVSWKLVTEYAMETK
                     CDDVLLLLGMYLEFQYSFEMCLKCIKKEQPSHYKYHEKHYANAAIFADSKNQKTICQQA
                     VDTVLAKKRVDSLQLTREQMLTNRFNDLLDRMDIMFGSTGSADIEEWMAGVAWLHCLLP
                     KMDSVVYDFLKCMVYNIPKKRYWLFKGPIDSGKTTLAAALLELCGGKALNVNLPLDRLN
                     FELGVAIDQFLVVFEDVKGTGGESRDLPSGQGINNLDNLRDYLDGSVKVNLEKKHLNKR
                     TQIFPPGIVTMNEYSVPKTLQARFVKQIDFRPKDYLKHCLERSEFLLEKRIIQSGIALL
                     LMLIWYRPVAEFAQSIQSRIVEWKERLDKEFSLSVYQKMKFNVAMGIGVLDWLRNSDDD
                     DEDSQENADKNEDGGEKNMEDSGHETGIDSQSQGSFQAPQSSQSVHDHNQPYHICRGFT
                     CFKKPPTPPPEPET"
     misc_feature    2159..2745
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2326..2343)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2553..2572
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2746..3345
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2746..2765)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3242..3262
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    3459..3492
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     LTR             3517..3697
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3723..3740)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3729..3953
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3999..4427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4086..4105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4296..4317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4436..4456)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4441..4770
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4621..4756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4683..4702
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4818..4865
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4884..5258
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    5388..5509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5425..5444)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5479..5498
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5558..5574)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5558..5574)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5571..5593)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5582..5598
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5606..5636)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5651..5672
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5789..5806)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5960..6548)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6040..6059)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6719..7579)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7342..7361
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7580..7684)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7759..7778)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7950..8329
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8331..8529
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8484..8504
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             8547..8727
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    8774..8899
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    9392..9625
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             9810..9854
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    10152..10269
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        10405..11583
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          10636..11574
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     11531..11551
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     11600..11619
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        11600..11618
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"

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