LOCUS       sequence-59196-e        6478 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 6478)
  AUTHORS   Ding Q, Lee YK, Schaefer EA, Peters DT, Veres A, Kim K, Kuperwasser 
            N, Motola DL, Meissner TB, Hendriks WT, Trevisan M, Gupta RM, Moisan
            A, Banks E, Friesen M, Schinzel RT, Xia F, Tang A, Xia Y, Figueroa 
            E, Wann A, Ahfeldt T, Daheron L, Zhang F, Rubin LL, Peng LF, Chung 
            RT, Musunuru K, Cowan CA
  TITLE     A TALEN Genome-Editing System for Generating Human Stem Cell-Based 
            Disease Models.
  JOURNAL   Cell Stem Cell. 2012 Dec 12. pii: S1934-5909(12)00645-5. doi: 
            10.1016/j.stem.2012.11.011.
  PUBMED    23246482
REFERENCE   2  (bases 1 to 6478)
  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. 2012 Dec 12. pii: S1934-5909(12)00645-5. doi: 
            10.1016/j.stem.2012.11.011."
COMMENT     Sequence Label: pTAL_GFP
FEATURES             Location/Qualifiers
     source          1..6478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..665
                     /label=chicken beta-actin promoter
     intron          666..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1739..1748
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1748..1813
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1820..1840
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2501..3088
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKV
                     MEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMER
                     YVEENQTRDKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             3104..3157
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(3158..3160,3161..3163,3164..3877)
                     /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(3158..3160,3161..3163,3164..3874)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3203..3224)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3464..3483)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3811..3832
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3920..4041
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3957..3976)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4011..4030
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4090..4106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4090..4106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4103..4125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4114..4130
                     /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(4138..4144,4145..4162,4163..4168))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4183..4204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4321..4338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4492..5080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4572..4591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5251..6042,6043..6111))
                     /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     5874..5893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6112..6216)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6295..6314)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
      841 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccat ctccagcctc ggggctgtcc
     1561 gcagggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagtgcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgcggccgc
     1741 caccatggac tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga
     1801 tgacgatgac aagatggccc ccaagaagaa gaggaaggtg ggcattcacg gggtgccgtc
     1861 tagagtagat ttgagaactt tgggatattc acagcagcag caggaaaaga tcaagcccaa
     1921 agtgaggtcg acagtcgcgc agcatcacga agcgctggtg ggtcatgggt ttacacatgc
     1981 ccacatcgta gccttgtcgc agcaccctgc agcccttggc acggtcgccg tcaagtacca
     2041 ggacatgatt gcggcgttgc cggaagccac acatgaggcg atcgtcggtg tggggaaaca
     2101 gtggagcgga gcccgagcgc ttgaggccct gttgacggtc gcgggagagc tgagagggcc
     2161 tccccttcag ctggacacgg gccagttgct gaagatcgcg aagcggggag gagtcacggc
     2221 ggtcgaggcg gtgcacgcgt ggcgcaatgc gctcacggga gcacccctca acctgaccga
     2281 gacgttagtc tagtcgtctc aggagtcaat cgtggcccag ctttcgaggc cggaccccgc
     2341 gctggccgca ctcactaatg atcatcttgt agcgctggcc tgcctcggcg gacgacccgc
     2401 cttggatgcg gtgaagaagg ggctcccgca cgcgcctgca ttgattaagc ggaccaacag
     2461 aaggattccc gagaggacat cacatcgagt ggcaggatcc caactagtca aaagtgaact
     2521 ggaggagaag aaatctgaac ttcgtcataa attgaaatat gtgcctcatg aatatattga
     2581 attaattgaa attgccagaa atcccactca ggatagaatc cttgaaatga aggtaatgga
     2641 attttttatg aaagtttatg gatatagagg tgaacatttg ggtggatcaa ggaaaccgga
     2701 cggagcaatt tatactgtcg gatctcctat tgattacggt gtgatcgtgg atactaaagc
     2761 ttatagcgga ggttataatc tgccaattgg ccaagcagat gaaatggagc gatatgtcga
     2821 agaaaatcaa acacgagaca aacatctcaa ccctaatgaa tggtggaaag tctatccatc
     2881 ttctgtaacg gaatttaagt ttttatttgt gagtggtcac tttaaaggaa actacaaagc
     2941 tcagcttaca cgattaaatc atatcactaa ttgtaatgga gctgttctta gtgtagaaga
     3001 gcttttaatt ggtggagaaa tgattaaagc cggcacatta accttagagg aagtaagacg
     3061 gaaatttaat aacggcgaga taaactttag gtccggcggc ggagagggca gaggaagtct
     3121 tctaacatgc ggtgacgtgg aggagaatcc cggcccaatg gtgagcaagg gcgaggagct
     3181 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     3241 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     3301 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     3361 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     3421 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     3481 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     3541 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     3601 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     3661 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     3721 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     3781 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     3841 cgggatcact ctcggcatgg acgagctgta caagtaactg cagcgcgggg atctcatgct
     3901 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3961 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4021 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     4081 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4141 catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga gctaactcac
     4201 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4261 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4321 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4381 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4441 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4501 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4561 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4621 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4681 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4741 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4801 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4861 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4921 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4981 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5041 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5101 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5161 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5221 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5281 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5341 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5401 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5461 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5521 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5581 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5641 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5701 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5761 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5821 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5881 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5941 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6001 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6061 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6121 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6181 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6241 tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc tcagtacaat
     6301 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     6361 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     6421 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatat
//
