LOCUS       sequence-36597-e        6447 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 6447)
  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 6447)
  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: JDS70
FEATURES             Location/Qualifiers
     source          1..6447
                     /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             1708..2295
                     /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             2311..2352
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2362..2379
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2402..2419)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2408..2632
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2678..3106
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2765..2784)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2975..2996
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3115..3135)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3120..3450
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3301..3436
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3363..3382
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3498..3545
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3564..3962
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4120..4241
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4157..4176)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4211..4230
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4290..4306)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4290..4306)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4303..4325)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4314..4330
                     /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(4338..4344,4345..4362,4363..4368))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4383..4404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4521..4538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4692..5280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4772..4791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5451..6242,6243..6311))
                     /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     6074..6093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6312..6416)
                     /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 cagagacgat
     1441 taatgcgtct cgctgacacc cgaacaggtg gtcgccattg cttctaacat cggaggacgg
     1501 ccagccttgg agtccatcgt agcccaattg tccaggcccg atcccgcgtt ggctgcgtta
     1561 acgaatgacc atctggtggc gttggcatgt cttggtggac gacccgcgct cgatgcagtc
     1621 aaaaagggtc tgcctcatgc tcccgcattg atcaaaagaa ccaaccggcg gattcccgag
     1681 agaacttccc atcgagtcgc gggatcccaa ctagtcaaaa gtgaactgga ggagaagaaa
     1741 tctgaacttc gtcataaatt gaaatatgtg cctcatgaat atattgaatt aattgaaatt
     1801 gccagaaatt ccactcagga tagaattctt gaaatgaagg taatggaatt ttttatgaaa
     1861 gtttatggat atagaggtaa acatttgggt ggatcaagga aaccggacgg agcaatttat
     1921 actgtcggat ctcctattga ttacggtgtg atcgtggata ctaaagctta tagcggaggt
     1981 tataatctgc caattggcca agcagatgaa atgcaacgat atgtcgaaga aaatcaaaca
     2041 cgaaacaaac atatcaaccc taatgaatgg tggaaagtct atccatcttc tgtaacggaa
     2101 tttaagtttt tatttgtgag tggtcacttt aaaggaaact acaaagctca gcttacacga
     2161 ttaaatcata tcactaattg taatggagct gttcttagtg tagaagagct tttaattggt
     2221 ggagaaatga ttaaagccgg cacattaacc ttagaggaag tcagacggaa atttaataac
     2281 ggcgagataa acttttaagg gcccttcgaa ggtaagccta tccctaaccc tctcctcggt
     2341 ctcgattcta cgcgtaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca
     2401 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2461 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2521 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2581 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2641 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     2701 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2761 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2821 gctctaaatc ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2881 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2941 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3001 acactcaacc ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc
     3061 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     3121 tgtgtcagtt agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc
     3181 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3241 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3301 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3361 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3421 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3481 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
     3541 acgacaaggt gaggaactaa accatggcca agcctttgtc tcaagaagaa tccaccctca
     3601 ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca
     3661 gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg
     3721 ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc
     3781 tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt
     3841 gtcgacaggt gcttctcgat ctgcatcctg ggatcaaagc gatagtgaag gacagtgatg
     3901 gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct
     3961 aagcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc
     4021 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4081 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4141 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4201 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4261 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4321 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4381 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4441 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4501 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4561 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4621 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4681 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4741 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4801 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4861 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     4921 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4981 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5041 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5101 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5161 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5221 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5281 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5341 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5401 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5461 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5521 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5581 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5641 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5701 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5761 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5821 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5881 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5941 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6001 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6061 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6121 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6181 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6241 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6301 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6361 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6421 tttccccgaa aagtgccacc tgacgtc
//
