LOCUS       sequence-8852-ex        4999 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 4999)
  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 4999)
  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 F3bbs kan NNNTGGGCGk
FEATURES             Location/Qualifiers
     source          1..4999
                     /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(974..1768)
                     /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(1085..1104)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1695..1714
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      2060..2515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2147..2166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2357..2378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2541..2645
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2646..2714,2715..3506)
                     /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(2864..2883)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3677..4265
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4166..4185
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4419..4436
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4451..4591
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4517..4536)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4677..4699
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4693..4884)
                     /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 ctgcgatcgc cgcttttctc gctcggatga gcttacccgc catatccgca
      721 tccataccgg tcagaagccc ttccagtgtc gaatctgtat gcgtaacttc agtcgtagtg
      781 accaccttac cacccacatc cgcacccaca caggcgagaa gccttttgca tgcgacatct
      841 gcgggtcttc tgagtgagga tccaaacgcc cggcggcaac cgagcgttct gaacaaatcc
      901 agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagcga gctctcgaac
      961 cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     1021 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
     1081 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     1141 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     1201 cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca
     1261 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
     1321 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
     1381 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
     1441 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
     1501 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
     1561 gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct
     1621 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
     1681 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
     1741 ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag
     1801 atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc
     1861 agggcttccc aaccttacca gagggcgccc cagctggcaa ttccgacgga tcctaagtaa
     1921 gaagacgacg ggtgagaaat aatctagaga ctagaaaaag gccgacaagt cccgctccgc
     1981 tgaagatcct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     2041 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2101 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2161 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2221 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2281 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2341 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2401 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2461 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     2521 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2581 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2641 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2701 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2761 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2821 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2881 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2941 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3001 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3061 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3121 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3181 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3241 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3301 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3361 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3421 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3481 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3541 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3601 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3661 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3721 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3781 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3841 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3901 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3961 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4021 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4081 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4141 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4201 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4261 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4321 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4381 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4441 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4501 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     4561 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     4621 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4681 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     4741 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     4801 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     4861 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4921 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4981 tgcccggtta ctggaacgg
//
