LOCUS       sequence-410382-        6804 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6804)
  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 6804)
  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-2A-Puro
FEATURES             Location/Qualifiers
     source          1..6804
                     /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..1724)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1008..1010,1011..1013,1014..1724)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /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"
     CDS             1851..2450
                     /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(1851..1870)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2347..2367
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3237..3256)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3238..3256)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3263..3280)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3263..3279)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3272..3294)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3420..3848
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3507..3526)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3717..3738
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4192..4986
                     /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(4246..4265)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4856..4875
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(5004..5072,5073..5864)
                     /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(5222..5241)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6035..6623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6524..6543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6777..6794
                     /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 caagtacagc ggtcgcgact
     1741 ctagagtcga cggttctggt aagcaaactt tgaattttga ccttcttaag cttgcgggag
     1801 acgtcgagtc caaccccggg cccgcatgca agcttcagct gaagcttacc atgaccgagt
     1861 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     1921 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     1981 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     2041 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     2101 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     2161 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     2221 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     2281 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     2341 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     2401 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga ggcgcgccgg
     2461 tgcctgccct tctaggaatg ggggacaggg ggaggggagg agctagggaa agaaaacctg
     2521 gagtttgtgc cagggttttt gggattaagt tcttcattca ctaaggaagg aattgggaac
     2581 acaaagggtg ggggcagggg agtttggggc aactggttgg agggaaggtg aagttcaatg
     2641 atgctcttga ttttaatccc acatcatgta tcactttttt cttaaataaa gaagcctggg
     2701 acacagtaga tagacacact tatcttggtt tgtccttcag ttactgaggt ggggatggga
     2761 atatccaatg ctcataccca agtgaccctg aaactaaggt gccatttaca ctccttaagg
     2821 tcacacaaca tcagagggag agctgggatt gcagccaagt ttatttgtac agggccctgt
     2881 gataggctag ttcccaaaag cctgtgatgc aagaactttt gcccatagac tcagtcacca
     2941 tgtagctgtt acctgttcag agctggcttt ttgctttccc accctactct ggaattctta
     3001 aatggcttta tacttagaaa tcatcttatt tctgttgaac ctagatcacc ccaaccagaa
     3061 acttctatta atactttgtg ctttcttgat accagggtct atttggtttc cacttaaggt
     3121 ttttgcatac tctgcccata agtgactcat tagttacggc cggccaaaag ggcgaattct
     3181 gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc aattcgccct
     3241 atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     3301 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     3361 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     3421 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3481 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3541 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3601 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3661 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3721 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3781 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3841 cgcgaatttt aacaaaattc agggcgcaag ggctgctaaa ggaagcggaa cacgtagaaa
     3901 gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca
     3961 agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag
     4021 ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct
     4081 ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga
     4141 tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     4201 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4261 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4321 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4381 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4441 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4501 tcatcccacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4561 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4621 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4681 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4741 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4801 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4861 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4921 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4981 ttctgaattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5041 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5101 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5161 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5221 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5281 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5341 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5401 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5461 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5521 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5581 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     5641 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5701 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     5761 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5821 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5881 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5941 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6001 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6061 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6121 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6181 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6241 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6301 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6361 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6421 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6481 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6541 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6601 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6661 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6721 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     6781 agtcagtgag cgaggaagcg gaag
//
