Basic Vector Information
Ef1a_Large T-antigen_Ires_Puro vector Vector Map
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
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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