LOCUS       sequence-8849-ex        4997 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 4997)
  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 4997)
  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: 1352wUV2 F1bbs kan GCGTGGNNN
FEATURES             Location/Qualifiers
     source          1..4997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    50..66
                     /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)."
     primer_bind     55..77
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             306..575
                     /codon_start=1
                     /gene="E. coli rpoZ"
                     /product="DNA-directed RNA polymerase subunit omega"
                     /label=RNAP omega
                     /note="promotes RNA polymerase assembly"
                     /translation="MARVTVQDAVEKIGNRFDLVLVAARRARQMQVGGKDPLVPEENDK
                     TTVIALREIEEGLINNQILDVRERQEQQEQEAAELQAVTAIAEGR"
     CDS             585..608
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(805..1599)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(916..935)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1526..1545
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      2058..2513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2145..2164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2355..2376
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2539..2643
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2644..2712,2713..3504)
                     /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     complement(2862..2881)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3675..4263
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4164..4183
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4417..4434
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4449..4589
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4515..4534)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4675..4697
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4691..4882)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gaattccggg ctttacactt tatgcttccg gctcgcatta tgtgtcgact tgtgagcgga
       61 taacaatttc acacaggaaa cagctatgcc tcaacagcag caaatgcaac ctcccaattc
      121 aagtgcggac aacaaccctt tgcaacagca atcatcacaa aataccgtac caaacgtcct
      181 caaccaaatt aaccaaatct tttctccaga ggagcaacgc agcttattac aagaagccat
      241 cgaaacctgc aagaattttg aaaaaacaca attgtaagtc tcggctccaa gaaggagata
      301 tacccatggc acgcgtaact gttcaggacg ctgtagagaa aattggtaac cgttttgacc
      361 tggtactggt cgccgcgcgt cgcgctcgtc agatgcaggt aggcggaaag gacccgctcg
      421 taccggaaga aaacgataaa accactgtaa tcgcgctgcg cgaaatcgaa gaaggtctga
      481 tcaacaacca gatcctcgac gttcgcgaac gccaggaaca gcaagagcag gaagccgctg
      541 aattacaagc cgttaccgct attgctgaag gtcgtgcggc cgcggactac aaggatgacg
      601 acgacaagtt ccggaccggt tccaagacac ccccccatgg tacccgccca tatgcttgcc
      661 ctgtcgagtc ctgtggtctt ctaagtgagg atccaaacgc ccggcggcaa ccgagcgttc
      721 tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcg
      781 agctctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
      841 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
      901 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
      961 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     1021 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     1081 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     1141 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     1201 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     1261 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     1321 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     1381 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     1441 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     1501 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     1561 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     1621 ctcttgatca gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca
     1681 gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccgacgg
     1741 atcctaagta agaagacacc ggccagaagc ccttccagtg tcgcatctgc atgcgtaact
     1801 tcagtcgctc cagccacctg accacccaca tcaggaccca cacaggcgag aagccttttg
     1861 catgcgacat ttgtgggagg aagtttgcca ggagtgatga acgcaagagg cataccaaaa
     1921 tccatacagg tgaaaagtaa tctagagact agaaaaaggc cgacaagtcc cgctccgctg
     1981 aagatcctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2041 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2101 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2161 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2221 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2281 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2341 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2401 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2461 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     2521 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2581 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2641 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2701 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2761 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2821 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2881 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2941 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3001 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3061 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3121 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3181 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3241 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3301 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3361 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3421 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3481 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3541 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3601 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3661 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3721 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3781 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3841 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3901 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3961 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4021 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4081 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4141 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4201 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4261 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4321 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4381 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4441 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4501 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4561 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4621 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4681 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     4741 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     4801 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     4861 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     4921 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     4981 cccggttact ggaacgg
//
