LOCUS       sequence-8851-ex        5026 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 5026)
  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 5026)
  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-Flag-TTGZFP-FokI-DD
FEATURES             Location/Qualifiers
     source          1..5026
                     /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..183
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             451..1038
                     /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
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQD
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     polyA_signal    1071..1205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1095..1114)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1149..1168
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1322..1342)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1322..1340)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1361..1377)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1361..1377)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1374..1396)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1385..1401
                     /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(1409..1415,1416..1433,1434..1439))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1454..1475
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1592..1609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1763..2351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1843..1862)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2522..3313,3314..3382))
                     /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     3145..3164
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3383..3487)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3513..3968)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3650..3671)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3862..3881
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4042..5026
                     /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 taaggtagac
      121 tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac
      181 aagggtaccc gcccatatgc ttgccctgtc gagtcctgtg accgccgctt cagccagaag
      241 gggcacctca cccgccatat ccggattcac acaggccaga agcccttcca gtgtcgcatc
      301 tgcatgagga acttcagccg cagcgacgcg ctcacccgcc acatccgcac ccacacaggc
      361 gagaagcctt ttgcatgcga catctgcggc aggaagttcg cgcgcagcga cgcgctcagg
      421 aagcacacca aaatccatac aggtggatcc caactagtca aaagtgaact ggaggagaag
      481 aaatctgaac ttcgtcataa attgaaatat gtgcctcatg aatatattga attaattgaa
      541 attgccagaa attccactca ggatagaatt cttgaaatga aggtaatgga attttttatg
      601 aaagtttatg gatatagagg taaacatttg ggtggatcaa ggaaaccgga cggagcaatt
      661 tatactgtcg gatctcctat tgattacggt gtgatcgtgg atactaaagc ttatagcgga
      721 ggttataatc tgccaattgg ccaagcagat gaaatgcaag actatgtcga agaaaatcaa
      781 acacgaaaca aacatatcaa ccctaatgaa tggtggaaag tctatccatc ttctgtaacg
      841 gaatttaagt ttttatttgt gagtggtcac tttaaaggaa actacaaagc tcagcttaca
      901 cgattaaatc atatcactaa ttgtaatgga gctgttctta gtgtagaaga gcttttaatt
      961 ggtggagaaa tgattaaagc cggcacatta accttagagg aagtgagacg gaaatttaat
     1021 aacggcgaga taaactttta atctagaact atagtgagtc gtattacgta gatccagaca
     1081 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     1141 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     1201 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     1261 ttttttaatt cgcggccgcg gcgccaatgc attgggcccg gtacgtaccc agcttttgtt
     1321 ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     1381 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     1441 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     1501 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     1561 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     1621 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     1681 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     1741 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     1801 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     1861 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     1921 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     1981 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2041 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2101 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2161 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     2221 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2281 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2341 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     2401 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     2461 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     2521 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     2581 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     2641 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     2701 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     2761 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     2821 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     2881 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     2941 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     3001 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     3061 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     3121 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     3181 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     3241 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     3301 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     3361 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     3421 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     3481 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     3541 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     3601 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     3661 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     3721 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     3781 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     3841 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     3901 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     3961 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     4021 ctcttcgcta ttacgccagt cgaccatagc caattcaata tggcgtatat ggactcatgc
     4081 caattcaata tggtggatct ggacctgtgc caattcaata tggcgtatat ggactcgtgc
     4141 caattcaata tggtggatct ggaccccagc caattcaata tggcggactt ggcaccatgc
     4201 caattcaata tggcggactt ggcactgtgc caactgggga ggggtctact tggcacggtg
     4261 ccaagtttga ggaggggtct tggccctgtg ccaagtccgc catattgaat tggcatggtg
     4321 ccaataatgg cggccatatt ggctatatgc caggatcaat atataggcaa tatccaatat
     4381 ggccctatgc caatatggct attggccagg ttcaatacta tgtattggcc ctatgccata
     4441 tagtattcca tatatgggtt ttcctattga cgtagatagc ccctcccaat gggcggtccc
     4501 atataccata tatggggctt cctaataccg cccatagcca ctcccccatt gacgtcaatg
     4561 gtctctatat atggtctttc ctattgacgt catatgggcg gtcctattga cgtatatggc
     4621 gcctccccca ttgacgtcaa ttacggtaaa tggcccgcct ggctcaatgc ccattgacgt
     4681 caataggacc acccaccatt gacgtcaatg ggatggctca ttgcccattc atatccgttc
     4741 tcacgccccc tattgacgtc aatgacggta aatggcccac ttggcagtac atcaatatct
     4801 attaatagta acttggcaag tacattacta ttggaaggac gccagggtac attggcagta
     4861 ctcccattga cgtcaatggc ggtaaatggc ccgcgatggc tgccaagtac atccccattg
     4921 acgtcaatgg ggaggggcaa tgacgcaaat gggcgttcca ttgacgtaaa tgggcggtag
     4981 gcgtgcctaa tgggaggtct atataagcaa tgctcgttta gggaac
//
