LOCUS       sequence-36592-e        7854 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 7854)
  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 7854)
  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"; doi: "10.1038/nbt"; 
            journalName: "Nat Biotechnol"; date: "2011-08-5- 5"; volume: "29"; 
            issue: "8"; pages: "697-8"
COMMENT     Sequence Label: TALEN1258
FEATURES             Location/Qualifiers
     source          1..7854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             907..972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             979..999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3115..3702
                     /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             3718..3759
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3769..3786
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3809..3826)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3815..4039
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4085..4513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4172..4191)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4382..4403
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4522..4542)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4527..4857
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4708..4843
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4770..4789
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4905..4952
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4971..5369
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5527..5648
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5564..5583)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5618..5637
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5697..5713)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5697..5713)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5710..5732)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5721..5737
                     /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(5745..5751,5752..5769,5770..5775))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5790..5811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5928..5945)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6099..6687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6179..6198)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6858..7649,7650..7718))
                     /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     7481..7500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7719..7823)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 accatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
      961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gcattcaccg cggggtacct
     1021 atggtggact tgaggacact cggttattcg caacagcaac aggagaaaat caagcctaag
     1081 gtcaggagca ccgtcgcgca acaccacgag gcgcttgtgg ggcatggctt cactcatgcg
     1141 catattgtcg cgctttcaca gcaccctgcg gcgcttggga cggtggctgt caaataccaa
     1201 gatatgattg cggccctgcc cgaagccacg cacgaggcaa ttgtaggggt cggtaaacag
     1261 tggtcgggag cgcgagcact tgaggcgctg ctgactgtgg cgggtgagct tagggggcct
     1321 ccgctccagc tcgacaccgg gcagctgctg aagatcgcga agagaggggg agtaacagcg
     1381 gtagaggcag tgcacgcctg gcgcaatgcg ctcaccgggg cccccttgaa cctgacccca
     1441 gaccaggtag tcgcaatcgc gtcacatgac gggggaaagc aagccctgga aaccgtgcaa
     1501 aggttgttgc cggtcctttg tcaagaccac ggccttacac cggagcaagt cgtggccatt
     1561 gcaaataata acggtggcaa acaggctctt gagacggttc agagacttct cccagttctc
     1621 tgtcaagccc acgggctgac tcccgatcaa gttgtagcga ttgcgtccaa cggtggaggg
     1681 aaacaagcat tggagactgt ccaacggctc cttcccgtgt tgtgtcaagc ccacggtttg
     1741 acgcctgcac aagtggtcgc catcgccagc catgatggcg gtaagcaggc gctggaaaca
     1801 gtacagcgcc tgctgcctgt actgtgccag gatcatggac tgaccccaga ccaggtagtc
     1861 gcaatcgcgt cacatgacgg gggaaagcaa gccctggaaa ccgtgcaaag gttgttgccg
     1921 gtcctttgtc aagaccacgg ccttacaccg gagcaagtcg tggccattgc aagcaacatc
     1981 ggtggcaaac aggctcttga gacggttcag agacttctcc cagttctctg tcaagcccac
     2041 gggctgactc ccgatcaagt tgtagcgatt gcgtcgaaca ttggagggaa acaagcattg
     2101 gagactgtcc aacggctcct tcccgtgttg tgtcaagccc acggtttgac gcctgcacaa
     2161 gtggtcgcca tcgcctcgaa tggcggcggt aagcaggcgc tggaaacagt acagcgcctg
     2221 ctgcctgtac tgtgccagga tcatggactg accccagacc aggtagtcgc aatcgcgtcg
     2281 aacattgggg gaaagcaagc cctggaaacc gtgcaaaggt tgttgccggt cctttgtcaa
     2341 gaccacggcc ttacaccgga gcaagtcgtg gccattgcaa ataataacgg tggcaaacag
     2401 gctcttgaga cggttcagag acttctccca gttctctgtc aagcccacgg gctgactccc
     2461 gatcaagttg tagcgattgc gtcgcatgac ggagggaaac aagcattgga gactgtccaa
     2521 cggctccttc ccgtgttgtg tcaagcccac ggtttgacgc ctgcacaagt ggtcgccatc
     2581 gcctcgaatg gcggcggtaa gcaggcgctg gaaacagtac agcgcctgct gcctgtactg
     2641 tgccaggatc atggactgac cccagaccag gtagtcgcaa tcgcgtcaaa cggaggggga
     2701 aagcaagccc tggaaaccgt gcaaaggttg ttgccggtcc tttgtcaaga ccacggcctt
     2761 acaccggagc aagtcgtggc cattgcatcc cacgacggtg gcaaacaggc tcttgagacg
     2821 gttcagagac ttctcccagt tctctgtcaa gcccacgggc tgacacccga acaggtggtc
     2881 gccattgctt ctaatggggg aggacggcca gccttggagt ccatcgtagc ccaattgtcc
     2941 aggcccgatc ccgcgttggc tgcgttaacg aatgaccatc tggtggcgtt ggcatgtctt
     3001 ggtggacgac ccgcgctcga tgcagtcaaa aagggtctgc ctcatgctcc cgcattgatc
     3061 aaaagaacca accggcggat tcccgagaga acttcccatc gagtcgcggg atcccaacta
     3121 gtcaaaagtg aactggagga gaagaaatct gaacttcgtc ataaattgaa atatgtgcct
     3181 catgaatata ttgaattaat tgaaattgcc agaaattcca ctcaggatag aattcttgaa
     3241 atgaaggtaa tggaattttt tatgaaagtt tatggatata gaggtaaaca tttgggtgga
     3301 tcaaggaaac cggacggagc aatttatact gtcggatctc ctattgatta cggtgtgatc
     3361 gtggatacta aagcttatag cggaggttat aatctgccaa ttggccaagc agatgaaatg
     3421 caacgatatg tcgaagaaaa tcaaacacga aacaaacata tcaaccctaa tgaatggtgg
     3481 aaagtctatc catcttctgt aacggaattt aagtttttat ttgtgagtgg tcactttaaa
     3541 ggaaactaca aagctcagct tacacgatta aatcatatca ctaattgtaa tggagctgtt
     3601 cttagtgtag aagagctttt aattggtgga gaaatgatta aagccggcac attaacctta
     3661 gaggaagtca gacggaaatt taataacggc gagataaact tttaagggcc cttcgaaggt
     3721 aagcctatcc ctaaccctct cctcggtctc gattctacgc gtaccggtca tcatcaccat
     3781 caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     3841 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     3901 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     3961 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     4021 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     4081 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4141 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4201 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt agggttccga
     4261 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4321 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4381 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     4441 ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     4501 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     4561 ccccaggcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     4621 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4681 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4741 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     4801 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     4861 ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat
     4921 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagc
     4981 ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc
     5041 ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca
     5101 ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca
     5161 ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca
     5221 ggggcatctt gagcccctgc ggacggtgtc gacaggtgct tctcgatctg catcctggga
     5281 tcaaagcgat agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc
     5341 tgccctctgg ttatgtgtgg gagggctaag cacttcgtgg ccgaggagca ggactgacac
     5401 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5461 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5521 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5581 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5641 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5701 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5761 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5821 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5881 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5941 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6001 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6061 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6121 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6181 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6241 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     6301 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6361 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6421 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6481 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     6541 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6601 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6661 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6721 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6781 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6841 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6901 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6961 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7021 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7081 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7141 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7201 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7261 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7321 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7381 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7441 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7501 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7561 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7621 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7681 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7741 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7801 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
