LOCUS       sequence-12722-e        4894 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 4894)
  AUTHORS   Zou J, Maeder ML, Mali P, Pruett-Miller SM, Thibodeau-Beganny S, 
            Chou BK, Chen G, Ye Z, Park IH, Daley GQ, Porteus MH, Joung JK, 
            Cheng L
  TITLE     Gene targeting of a disease-related gene in human induced 
            pluripotent stem and embryonic stem cells.
  JOURNAL   Cell Stem Cell. 2009 Jul 2. 5(1):97-110.
  PUBMED    19540188
REFERENCE   2  (bases 1 to 4894)
  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: "Cell Stem 
            Cell. 2009 Jul 2. 5(1):97-110."
COMMENT     Sequence Label: tGFP
FEATURES             Location/Qualifiers
     source          1..4894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     677..693
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     677..693
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(704..725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(965..984)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1312..1333
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1418..1992,1993..1995,1996..2004)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1585..1602)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1812..1831
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2005..2604
                     /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(2005..2024)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2501..2521
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2660..2676)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2661..2677)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2706..2726)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2706..2724)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2745..2761)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2745..2761)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2758..2780)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2769..2785
                     /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(join(2793..2799,2800..2817,2818..2823))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2838..2859
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2976..2993)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3147..3735)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3227..3246)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3906..4697,4698..4766))
                     /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     4529..4548
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4767..4871)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgctcta gaactagtgg atccgtgccc atcctggtcg agctggacgg
      721 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      781 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      841 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
      901 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
      961 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1021 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1081 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1141 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1201 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1261 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1321 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag
     1381 cggccgcgtc gacgggcccg cggaattccg cccccccccc ctctccctcc ccccccccta
     1441 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     1501 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     1561 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     1621 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     1681 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     1741 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     1801 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     1861 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     1921 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     1981 cacgatgata atatggccac aaccatgacc gagtacaagc ccacggtgcg cctcgccacc
     2041 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     2101 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     2161 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     2221 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     2281 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     2341 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     2401 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     2461 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     2521 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     2581 atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     2641 agcgcacgac cccatgcatc gataccgtcg acctcgaggg ggggcccggt acccagcttt
     2701 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     2761 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     2821 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     2881 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2941 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3001 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3061 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3121 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3181 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3241 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3301 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3361 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3421 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3481 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3541 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     3601 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3661 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3721 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3781 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3841 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3901 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3961 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4021 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4081 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4141 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4201 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4261 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     4321 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     4381 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     4441 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4501 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4561 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4621 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4681 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4741 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4801 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4861 ggggttccgc gcacatttcc ccgaaaagtg ccac
//
