LOCUS       sequence-496842-        8057 bp    DNA     circular SYN 09-JAN-2026
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8057)
  AUTHORS   Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR
  TITLE     Targeted gene disruption in somatic zebrafish cells using engineered
            TALENs.
  JOURNAL   Nat Biotechnol. 2011 Aug 5;29(8):697-8. doi: 10.1038/nbt.1934.
  PUBMED    21822241
REFERENCE   2  (bases 1 to 8057)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 9, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt"; 
            journalName: "Nat Biotechnol"; date: "2011-08-5- 5"; volume: "29"; 
            issue: "8"; pages: "697-8"
COMMENT     Sequence Label: TAL3195
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             673..738
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             745..765
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3085..3672
                     /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="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             3688..3729
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3739..3756
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3779..3796)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3785..4009
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4055..4483
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4142..4161)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4352..4373
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4492..4512)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4497..4826
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4677..4812
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4739..4758
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4874..4921
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4940..5338
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5496..5617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5533..5552)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5587..5606
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5666..5682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5666..5682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5679..5701)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5690..5706
                     /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(5714..5720,5721..5738,5739..5744))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5759..5780
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5897..5914)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6068..6656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6148..6167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6827..7618,7619..7687))
                     /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     7450..7469
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7688..7792)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7867..7886)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcacca tggactacaa agaccatgac ggtgattata aagatcatga catcgattac
      721 aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggcat tcaccgcggg
      781 gtacctatgg tggacttgag gacactcggt tattcgcaac agcaacagga gaaaatcaag
      841 cctaaggtca ggagcaccgt cgcgcaacac cacgaggcgc ttgtggggca tggcttcact
      901 catgcgcata ttgtcgcgct ttcacagcac cctgcggcgc ttgggacggt ggctgtcaaa
      961 taccaagata tgattgcggc cctgcccgaa gccacgcacg aggcaattgt aggggtcggt
     1021 aaacagtggt cgggagcgcg agcacttgag gcgctgctga ctgtggcggg tgagcttagg
     1081 gggcctccgc tccagctcga caccgggcag ctgctgaaga tcgcgaagag agggggagta
     1141 acagcggtag aggcagtgca cgcctggcgc aatgcgctca ccggggcccc cttgaacctg
     1201 accccagacc aggtagtcgc aatcgcgtcg aacattgggg gaaagcaagc cctggaaacc
     1261 gtgcaaaggt tgttgccggt cctttgtcaa gaccacggcc ttacaccgga gcaagtcgtg
     1321 gccattgcaa gcaacatcgg tggcaaacag gctcttgaga cggttcagag acttctccca
     1381 gttctctgtc aagcccacgg gctgactccc gatcaagttg tagcgattgc gtcgaacatt
     1441 ggagggaaac aagcattgga gactgtccaa cggctccttc ccgtgttgtg tcaagcccac
     1501 ggtttgacgc ctgcacaagt ggtcgccatc gccagccatg atggcggtaa gcaggcgctg
     1561 gaaacagtac agcgcctgct gcctgtactg tgccaggatc atggactgac cccagaccag
     1621 gtagtcgcaa tcgcgtcaca tgacggggga aagcaagccc tggaaaccgt gcaaaggttg
     1681 ttgccggtcc tttgtcaaga ccacggcctt acaccggagc aagtcgtggc cattgcatcc
     1741 cacgacggtg gcaaacaggc tcttgagacg gttcagagac ttctcccagt tctctgtcaa
     1801 gcccacgggc tgactcccga tcaagttgta gcgattgcga ataacaatgg agggaaacaa
     1861 gcattggaga ctgtccaacg gctccttccc gtgttgtgtc aagcccacgg tttgacgcct
     1921 gcacaagtgg tcgccatcgc ctcgaatggc ggcggtaagc aggcgctgga aacagtacag
     1981 cgcctgctgc ctgtactgtg ccaggatcat ggactgaccc cagaccaggt agtcgcaatc
     2041 gcgtcaaacg gagggggaaa gcaagccctg gaaaccgtgc aaaggttgtt gccggtcctt
     2101 tgtcaagacc acggccttac accggagcaa gtcgtggcca ttgcaagcaa tgggggtggc
     2161 aaacaggctc ttgagacggt tcagagactt ctcccagttc tctgtcaagc ccacgggctg
     2221 actcccgatc aagttgtagc gattgcgtcc aacggtggag ggaaacaagc attggagact
     2281 gtccaacggc tccttcccgt gttgtgtcaa gcccacggtt tgacgcctgc acaagtggtc
     2341 gccatcgcca gccatgatgg cggtaagcag gcgctggaaa cagtacagcg cctgctgcct
     2401 gtactgtgcc aggatcatgg actgacccca gaccaggtag tcgcaatcgc gtcgaacatt
     2461 gggggaaagc aagccctgga aaccgtgcaa aggttgttgc cggtcctttg tcaagaccac
     2521 ggccttacac cggagcaagt cgtggccatt gcaagcaaca tcggtggcaa acaggctctt
     2581 gagacggttc agagacttct cccagttctc tgtcaagccc acgggctgac tcccgatcaa
     2641 gttgtagcga ttgcgaataa caatggaggg aaacaagcat tggagactgt ccaacggctc
     2701 cttcccgtgt tgtgtcaagc ccacggtttg acgcctgcac aagtggtcgc catcgccaac
     2761 aacaacggcg gtaagcaggc gctggaaaca gtacagcgcc tgctgcctgt actgtgccag
     2821 gatcatggac tgacacccga acaggtggtc gccattgctt cccacgacgg aggacggcca
     2881 gccttggagt ccatcgtagc ccaattgtcc aggcccgatc ccgcgttggc tgcgttaacg
     2941 aatgaccatc tggtggcgtt ggcatgtctt ggtggacgac ccgcgctcga tgcagtcaaa
     3001 aagggtctgc ctcatgctcc cgcattgatc aaaagaacca accggcggat tcccgagaga
     3061 acttcccatc gagtcgcggg atcccaacta gtcaaaagtg aactggagga gaagaaatct
     3121 gaacttcgtc ataaattgaa atatgtgcct catgaatata ttgaattaat tgaaattgcc
     3181 agaaattcca ctcaggatag aattcttgaa atgaaggtaa tggaattttt tatgaaagtt
     3241 tatggatata gaggtaaaca tttgggtgga tcaaggaaac cggacggagc aatttatact
     3301 gtcggatctc ctattgatta cggtgtgatc gtggatacta aagcttatag cggaggttat
     3361 aatctgccaa ttggccaagc agatgaaatg caacgatatg tcgaagaaaa tcaaacacga
     3421 aacaaacata tcaaccctaa tgaatggtgg aaagtctatc catcttctgt aacggaattt
     3481 aagtttttat ttgtgagtgg tcactttaaa ggaaactaca aagctcagct tacacgatta
     3541 aatcatatca ctaattgtaa tggagctgtt cttagtgtag aagagctttt aattggtgga
     3601 gaaatgatta aagccggcac attaacctta gaggaagtca gacggaaatt taataacggc
     3661 gagataaact tttaagggcc cttcgaaggt aagcctatcc ctaaccctct cctcggtctc
     3721 gattctacgc gtaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc
     3781 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3841 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3901 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3961 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     4021 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     4081 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     4141 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4201 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4261 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4321 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4381 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4441 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     4501 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4561 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4621 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4681 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4741 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     4801 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     4861 ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     4921 caaggtgagg aactaaacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga
     4981 aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc
     5041 agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg
     5101 accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac
     5161 ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg
     5221 acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca
     5281 gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaagc
     5341 acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct
     5401 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5461 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5521 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5581 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5641 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5701 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5761 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5821 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5881 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5941 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6001 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6061 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6121 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6181 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6241 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6301 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6361 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6421 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6481 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6541 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6601 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6661 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6721 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6781 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6841 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6901 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6961 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7021 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7081 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7141 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7201 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7261 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7321 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7381 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7441 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7501 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7561 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7621 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7681 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7741 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7801 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     7861 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     7921 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     7981 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     8041 ggccagatat acgcgtt
//
