LOCUS       sequence-8850-ex        5056 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 5056)
  AUTHORS   Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe SA
  TITLE     Targeted gene inactivation in zebrafish using engineered zinc-finger
            nucleases.
  JOURNAL   Nat Biotechnol. 2008 Jun . 26(6):695-701.
  PUBMED    18500337
REFERENCE   2  (bases 1 to 5056)
  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: "Nat 
            Biotechnol. 2008 Jun . 26(6):695-701."
COMMENT     Sequence Label: pCS2-HA-GAAZFP-FokI-RR
FEATURES             Location/Qualifiers
     source          1..5056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             97..117
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             118..207
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             481..1068
                     /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
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQAREMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     polyA_signal    1101..1235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1125..1144)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1179..1198
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1352..1372)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1352..1370)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1391..1407)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1391..1407)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1404..1426)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1415..1431
                     /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(1439..1445,1446..1463,1464..1469))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1484..1505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1622..1639)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1793..2381)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1873..1892)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2552..3343,3344..3412))
                     /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     3175..3194
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3413..3517)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3543..3998)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3680..3701)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3892..3911
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4072..5056
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atctgccacc atggctccaa agaagaagcg taaggtatac
      121 ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcg
      181 tatccatatg acgttccaga ttacgctgct catggtaccc gcccatatgc ttgccctgtc
      241 gagtcctgtg accgccgctt cagccgtagc gacgcgctca cccgccatat ccggattcac
      301 acaggccaga agcccttcca gtgtcgcatc tgcatgagga acttcagctg tgcgcatcac
      361 ctcacccgcc acatccgcac ccacacaggc gagaagcctt ttgcatgcga catttgcggc
      421 aggaagttcg cgcagttgtc taacctcacc cgccatacca aaatccatac aggtggatcc
      481 caactagtca aaagtgaact ggaggagaag aaatctgaac ttcgtcataa attgaaatat
      541 gtgcctcatg aatatattga attaattgaa attgccagaa attccactca ggatagaatt
      601 cttgaaatga aggtaatgga attttttatg aaagtttatg gatatagagg taaacatttg
      661 ggtggatcaa ggaaaccgga cggagcaatt tatactgtcg gatctcctat tgattacggt
      721 gtgatcgtgg atactaaagc ttatagcgga ggttataatc tgccaattgg ccaagcacgt
      781 gaaatgcaac gatatgtcga agaaaatcaa acacgaaaca aacatatcaa ccctaatgaa
      841 tggtggaaag tctatccatc ttctgtaacg gaatttaagt ttttatttgt gagtggtcac
      901 tttaaaggaa actacaaagc tcagcttaca cgattaaatc atatcactaa ttgtaatgga
      961 gctgttctta gtgtagaaga gcttttaatt ggtggagaaa tgattaaagc cggcacatta
     1021 accttagagg aagtgagacg gaaatttaat aacggcgaga taaactttta atctagaact
     1081 atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa
     1141 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     1201 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     1261 tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcggccgcg gcgccaatgc
     1321 attgggcccg gtacgtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     1381 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1441 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1501 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1561 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1621 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1681 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1741 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1801 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1861 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1921 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1981 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2041 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2101 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2161 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2221 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2281 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2341 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2401 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2461 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2521 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     2581 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     2641 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     2701 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     2761 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     2821 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     2881 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     2941 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3001 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3061 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3121 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3181 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3241 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3301 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3361 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3421 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3481 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     3541 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3601 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3661 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3721 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3781 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3841 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3901 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3961 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
     4021 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagt cgaccatagc
     4081 caattcaata tggcgtatat ggactcatgc caattcaata tggtggatct ggacctgtgc
     4141 caattcaata tggcgtatat ggactcgtgc caattcaata tggtggatct ggaccccagc
     4201 caattcaata tggcggactt ggcaccatgc caattcaata tggcggactt ggcactgtgc
     4261 caactgggga ggggtctact tggcacggtg ccaagtttga ggaggggtct tggccctgtg
     4321 ccaagtccgc catattgaat tggcatggtg ccaataatgg cggccatatt ggctatatgc
     4381 caggatcaat atataggcaa tatccaatat ggccctatgc caatatggct attggccagg
     4441 ttcaatacta tgtattggcc ctatgccata tagtattcca tatatgggtt ttcctattga
     4501 cgtagatagc ccctcccaat gggcggtccc atataccata tatggggctt cctaataccg
     4561 cccatagcca ctcccccatt gacgtcaatg gtctctatat atggtctttc ctattgacgt
     4621 catatgggcg gtcctattga cgtatatggc gcctccccca ttgacgtcaa ttacggtaaa
     4681 tggcccgcct ggctcaatgc ccattgacgt caataggacc acccaccatt gacgtcaatg
     4741 ggatggctca ttgcccattc atatccgttc tcacgccccc tattgacgtc aatgacggta
     4801 aatggcccac ttggcagtac atcaatatct attaatagta acttggcaag tacattacta
     4861 ttggaaggac gccagggtac attggcagta ctcccattga cgtcaatggc ggtaaatggc
     4921 ccgcgatggc tgccaagtac atccccattg acgtcaatgg ggaggggcaa tgacgcaaat
     4981 gggcgttcca ttgacgtaaa tgggcggtag gcgtgcctaa tgggaggtct atataagcaa
     5041 tgctcgttta gggaac
//
