LOCUS       sequence-224391-        7958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Oct4 targeting donor vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7958)
  AUTHORS   Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, 
            Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X, 
            Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R
  TITLE     Genetic engineering of human pluripotent cells using TALE nucleases.
  JOURNAL   Nat Biotechnol. 2011 Jul 7. doi: 10.1038/nbt.1927.
  PUBMED    21738127
REFERENCE   2  (bases 1 to 7958)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2011 Jul 7. doi: 10.1038/nbt.1927."
COMMENT     Sequence Label: OCT4-eGFP-PGK-Puro
FEATURES             Location/Qualifiers
     source          1..7958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(1008..1010,1011..1013,1014..1727)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1008..1010,1011..1013,1014..1724)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1053..1074)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1314..1333)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1661..1682
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1758..1791
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1852..2351
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1887..1910)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2284..2303
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2391..2990
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2391..2410)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    3547..3580
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4391..4410)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4392..4410)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4417..4434)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4417..4433)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4426..4448)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4574..5002
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4661..4680)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4871..4892
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             5346..6140
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5400..5419)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6010..6029
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(6158..6226,6227..7018)
                     /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     complement(6376..6395)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7189..7777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7678..7697
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7931..7948
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgc ccttttcctg
      301 caggtcccac ctgcacagat atgcaaagca gaaaccctcg tgcaggcccg aaagagaaag
      361 cgaaccagta tcgagaaccg agtgagaggc aacctggaga atttgttcct gcagtgcccg
      421 aaacccacac tgcagcagat cagccacatc gcccagcagc ttgggctcga gaaggatgtg
      481 agtgccatgt ctctctgcgg gctccatctc tttcccctgt caccacctcg ctttccctag
      541 ctctggctcc tccaactgct ctagggctgt tggctttgga cagaatgtcc aagcagtcag
      601 gcctgtctca gctcattctc taatgtcctc ctctaactgc tctagggctg ttggctttgg
      661 atagaatgtc caagcagagt caggcccgtc tcagctcatt gtctaatgtc attctccttt
      721 ctgtcattca cttgcaggtg gtccgagtgt ggttctgtaa ccggcgccag aagggcaagc
      781 gatcaagcag cgactatgca caacgagagg attttgaggc tgctgggtct cctttctcag
      841 ggggaccagt gtcctttcct ctggccccag ggccccattt tggtacccca ggctatggga
      901 gccctcactt cactgcactg tactcctcgg tccctttccc tgagggggaa gcctttcccc
      961 ctgtctccgt caccactctg ggctctccca tgcattcaaa cgctagcatg gtgagcaagg
     1021 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1081 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1141 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1201 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1261 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1321 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1381 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1441 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1501 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1561 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1621 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1681 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagaa ttccgatcat
     1741 attcaataac ccttaatata acttcgtata atgtatgcta tacgaagtta ttaggtctga
     1801 agaggagttt acgtccagcc aagcttagga tctcgacctc gaaattctac cgggtagggg
     1861 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     1921 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccga
     1981 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     2041 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     2101 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     2161 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     2221 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     2281 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     2341 cgggcctttc gacctgcatc catctagatc tcgagcagct gaagcttacc atgaccgagt
     2401 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     2461 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     2521 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     2581 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     2641 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     2701 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     2761 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     2821 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     2881 cgccccacaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     2941 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc
     3001 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg atatcagatc
     3061 cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa aatggaagtt
     3121 tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt gaatggaagg
     3181 attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc tttactgaag
     3241 gctctttact attgctttat gataatgttt catagttgga tatcataatt taaacaagca
     3301 aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct atagatctct
     3361 cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact gtggtttcca
     3421 aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca catacacttc
     3481 attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag atccggaacc
     3541 cttaatataa cttcgtataa tgtatgctat acgaagttat taggtccctc gaagaggttc
     3601 actaggcgcg ccggtgcctg cccttctagg aatgggggac agggggaggg gaggagctag
     3661 ggaaagaaaa cctggagttt gtgccagggt ttttgggatt aagttcttca ttcactaagg
     3721 aaggaattgg gaacacaaag ggtgggggca ggggagtttg gggcaactgg ttggagggaa
     3781 ggtgaagttc aatgatgctc ttgattttaa tcccacatca tgtatcactt ttttcttaaa
     3841 taaagaagcc tgggacacag tagatagaca cacttatctt ggtttgtcct tcagttactg
     3901 aggtggggat gggaatatcc aatgctcata cccaagtgac cctgaaacta aggtgccatt
     3961 tacactcctt aaggtcacac aacatcagag ggagagctgg gattgcagcc aagtttattt
     4021 gtacagggcc ctgtgatagg ctagttccca aaagcctgtg atgcaagaac ttttgcccat
     4081 agactcagtc accatgtagc tgttacctgt tcagagctgg ctttttgctt tcccacccta
     4141 ctctggaatt cttaaatggc tttatactta gaaatcatct tatttctgtt gaacctagat
     4201 caccccaacc agaaacttct attaatactt tgtgctttct tgataccagg gtctatttgg
     4261 tttccactta aggtttttgc atactctgcc cataagtgac tcattagtta cggccggcca
     4321 aaagggcgaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg
     4381 gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
     4441 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4501 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4561 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4621 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4681 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4741 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4801 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4861 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4921 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4981 taacaaaaat ttaacgcgaa ttttaacaaa attcagggcg caagggctgc taaaggaagc
     5041 ggaacacgta gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact
     5101 gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc
     5161 ttacatggcg atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag
     5221 ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc
     5281 cgccaaggat ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt
     5341 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     5401 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     5461 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     5521 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     5581 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     5641 ggcaggatct cctgtcatcc caccttgctc ctgccgagaa agtatccatc atggctgatg
     5701 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     5761 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     5821 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     5881 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     5941 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     6001 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     6061 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     6121 ttcttgacga gttcttctga attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6181 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6241 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6301 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6361 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6421 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6481 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6541 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6601 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6661 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6721 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6781 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6841 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6901 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6961 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     7021 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7081 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7141 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7201 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7261 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7321 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7381 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7441 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7501 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7561 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7621 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7681 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7741 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7801 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     7861 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     7921 gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
