LOCUS       sequence-225145-        7999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  POC12397, MetClo assembly vector with F replication origin and 
            kanamycin resistance for BsaI-based MetClo, adaptor sequence type 
            p-a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7999)
  AUTHORS   Lin D, O'Callaghan CA
  TITLE     MetClo: methylase-assisted hierarchical DNA assembly using a single 
            type IIS restriction enzyme.
  JOURNAL   Nucleic Acids Res. 2018 Jul 9. pii: 5050631. doi: 
            10.1093/nar/gky596.
  PUBMED    29986052
REFERENCE   2  (bases 1 to 7999)
  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: "Nucleic 
            Acids Res. 2018 Jul 9. pii: 5050631. doi: 10.1093/nar/gky596."
COMMENT     Sequence Label: pMXBK_pFa
FEATURES             Location/Qualifiers
     source          1..7999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      220..808
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     709..728
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     962..979
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1096..1117
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1132..1137,1138..1155,1156..1162)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1170..1186
                     /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     1175..1197
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1194..1210
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1194..1210
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1206..1529
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1223..1279
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1280..1297)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1280..1296)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1289..1311)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1505..1524)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1692..1796
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1797..2591
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3406..3625
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3716..4471
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4474..4724
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5050..6225
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6225..7196
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7269..7742
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
ORIGIN
        1 cactctgtga cttggtctca ggagtgagac cggcctagca ggtgtgagtt tgtcagacca
       61 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
      121 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
      181 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
      241 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
      301 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
      361 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
      421 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
      481 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
      541 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
      601 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
      661 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
      721 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
      781 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
      841 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
      901 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
      961 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     1021 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     1081 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     1141 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     1201 cagctatgac catgattacg ccaagcttgc atgcctgcag gtcgactcta gaggatcccc
     1261 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     1321 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     1381 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
     1441 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     1501 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     1561 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgacaa
     1621 gtaccacctg gaacgccaaa ggtctcacgc taggttgaga ccaagtcacg aagtgtgcgg
     1681 ccgccttcgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1741 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatgg
     1801 ctaaaatgag aatatcaccg gaattgaaaa aactgatcga aaaataccgc tgcgtaaaag
     1861 atacggaagg aatgtctcct gctaaggtat ataagctggt gggagaaaat gaaaacctat
     1921 atttaaaaat gacggacagc cggtataaag ggaccaccta tgatgtggaa cgggaaaagg
     1981 acatgatgct atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac tttgaacggc
     2041 atgatggctg gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc tcggaagagt
     2101 atgaagatga acaaagccct gaaaagatta tcgagctgta tgcggagtgc atcaggctct
     2161 ttcactccat cgacatatcg gattgtccct atacgaatag cttagacagc cgcttagccg
     2221 aattggatta cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac tgggaagagg
     2281 acactccatt taaagatccg cgcgagctgt atgatttttt aaagacggaa aagcccgaag
     2341 aggaacttgt cttttcccac ggcgacctgg gagacagcaa catctttgtg aaagatggca
     2401 aagtaagtgg ctttattgat cttgggagaa gcggcagggc ggacaagtgg tatgacattg
     2461 ccttctgcgt ccggtcgctc agggaggata tcggggaaga acagtatgtc gagctatttt
     2521 ttgacttact ggggatcaag cctgattggg agaaaataaa atattatatt ttactggatg
     2581 aattgtttta ggagctggag cggcgtaacc gtcgcacagg aaggacagag aaagcgcgga
     2641 tctgggaagt gacggacaga acggtcagga cctggattgg ggaggcggtt gccgccgctg
     2701 ctgctgacgg tgtgacgttc tctgttccgg tcacaccaca tacgttccgc cattcctatg
     2761 cgatgcacat gctgtatgcc ggtataccgc tgaaagttct gcaaagcctg atgggacata
     2821 agtccatcag ttcaacggaa gtctacacga aggtttttgc gctggatgtg gctgcccggc
     2881 tccgggtgca gtttgcgatg ccggagtctg atgcggttgc gatgctgaaa caattatcct
     2941 gagaataaat gccttggcct ttatatggaa atgtggaact gagtggatat gctgtttttg
     3001 tctgttaaac agagaagctg gctgttatcc actgagaagc gaacgaaaca gtcgggaaaa
     3061 tctcccatta tcgtagagat ccgcattatt aatctcagga gcctgtgtag cgtttatagg
     3121 aagtagtgtt ctgtcatgat gcctgcaagc ggtaacgaaa acgatttgaa tatgccttca
     3181 ggaacaatag aaatcttcgt gcggtgttac gttgaagtgg agcggattat gtcagcaatg
     3241 gacagaacaa cctaatgaac acagaaccat gatgtggtct gtccttttac agccagtagt
     3301 gctcgccgca gtcgagcgac agggcgaagc cctcgagtga gcgaggaagc accagggaac
     3361 agcacttata tattctgctt acacacgatg cctgaaaaaa cttcccttgg ggttatccac
     3421 ttatccacgg ggatattttt ataattattt tttttatagt ttttagatct tcttttttag
     3481 agcgccttgt aggcctttat ccatgctggt tctagagaag gtgttgtgac aaattgccct
     3541 ttcagtgtga caaatcaccc tcaaatgaca gtcctgtctg tgacaaattg cccttaaccc
     3601 tgtgacaaat tgccctcaga agaagctgtt ttttcacaaa gttatccctg cttattgact
     3661 cttttttatt tagtgtgaca atctaaaaac ttgtcacact tcacatggat ctgtcatggc
     3721 ggaaacagcg gttatcaatc acaagaaacg taaaaatagc ccgcgaatcg tccagtcaaa
     3781 cgacctcact gaggcggcat atagtctctc ccgggatcaa aaacgtatgc tgtatctgtt
     3841 cgttgaccag atcagaaaat ctgatggcac cctacaggaa catgacggta tctgcgagat
     3901 ccatgttgct aaatatgctg aaatattcgg attgacctct gcggaagcca gtaaggatat
     3961 acggcaggca ttgaagagtt tcgcggggaa ggaagtggtt ttttatcgcc ctgaagagga
     4021 tgccggcgat gaaaaaggct atgaatcttt tccttggttt atcaaacgtg cgcacagtcc
     4081 atccagaggg ctttacagtg tacatatcaa cccatatctc attcccttct ttatcgggtt
     4141 acagaaccgg tttacgcagt ttcggcttag tgaaacaaaa gaaatcacca atccgtatgc
     4201 catgcgttta tacgaatccc tgtgtcagta tcgtaagccg gatggctcag gcatcgtgtc
     4261 tctgaaaatc gactggatca tagagcgtta ccagctgcct caaagttacc agcgtatgcc
     4321 tgacttccgc cgccgcttcc tgcaggtctg tgttaatgag atcaacagca gaactccaat
     4381 gcgcctctca tacattgaga aaaagaaagg ccgccagacg actcatatcg tattttcctt
     4441 ccgcgatatc acttccatga cgacaggata gtctgagggt tatctgtcac agatttgagg
     4501 gtggttcgtc acatttgttc tgacctactg agggtaattt gtcacagttt tgctgtttcc
     4561 ttcagcctgc atggattttc tcatactttt tgaactgtaa tttttaagga agccaaattt
     4621 gagggcagtt tgtcacagtt gatttccttc tctttccctt cgtcatgtga cctgatatcg
     4681 ggggttagtt cgtcatcatt gatgagggtt gattatcaca gtttattact ctgaattggc
     4741 tatccgcgtg tgtacctcta cctggagttt ttcccacggt ggatatttct tcttgcgctg
     4801 agcgtaagag ctatctgaca gaacagttct tctttgcttc ctcgccagtt cgctcgctat
     4861 gctcggttac acggctgcgg cgagcgctag tgataataag tgactgaggt atgtggtgtt
     4921 cttatctcct tttgtagtgt tgctcttatt ttaaacaact ttgcggtttt ttgatgactt
     4981 tgcgattttg ttgttgcttt gcagtaaatt gcaagattta ataaaaaaac gcaaagcaat
     5041 gattaaagga tgttcagaat gaaactcatg gaaacactta accagtgcat aaacgctggt
     5101 catgaaatga cgaaggctat cgccattgca cagtttaatg atgacagccc ggaagcgagg
     5161 aaaataaccc ggcgctggag aataggtgaa gcagcggatt tagttggggt ttcttctcag
     5221 gctatcagag atgccgagaa agcagggcga ctaccgcacc cggatatgga aattcgagga
     5281 cgggttgagc aacgtgttgg ttatacaatt gaacaaatta atcatatgcg tgatgtgttt
     5341 ggtacgcgat tgcgacgtgc tgaggacgta tttccaccgg tgatcggggt tgctgcccat
     5401 aaaggtggcg tttacaaaac ctcagtttct gttcatcttg ctcaggatct ggctctgaag
     5461 gggctacgtg ttttgctcgt ggaaggtaac gacccccagg gaacagcctc aatgtatcac
     5521 ggatgggtac cagatcttca tattcatgca gaagatactc tcctgccttt ctatcttggg
     5581 gaaaaggacg atgtcactta tgcaataaag cccacttgct ggccggggct tgacattatt
     5641 ccttcctgtc tggctctgca ccgtattgaa actgagttaa tgggcaaatt tgatgaaggt
     5701 aaactgccca ccgatccaca cctgatgctc cgactggcca ttgaaactgt tgctcatgac
     5761 tatgatgtca tagttattga cagcgcgcct aacctgggta tcggcacgat taatgtcgta
     5821 tgtgctgctg atgtgctgat tgttcccacg cctgctgagt tgtttgacta cacctccgca
     5881 ctgcagtttt tcgatatgct tcgtgatctg ctcaagaacg ttgatcttaa agggttcgag
     5941 cctgatgtac gtattttgct taccaaatac agcaatagta atggctctca gtccccgtgg
     6001 atggaggagc aaattcggga tgcctgggga agcatggttc taaaaaatgt tgtacgtgaa
     6061 acggatgaag ttggtaaagg tcagatccgg atgagaactg tttttgaaca ggccattgat
     6121 caacgcagtt caactggtgc ctggagaaat gctctttcta tttgggaacc tgtctgcaat
     6181 gaaattttcg atcgtctgat taaaccacgc tgggagatta gataatgaag cgtgcgcctg
     6241 ttattccaaa acatacgctc aatactcaac cggttgaaga tacttcgtta tcgacaccag
     6301 ctgccccgat ggtggattcg ttaattgcgc gcgtaggagt aatggctcgc ggtaatgcca
     6361 ttactttgcc tgtatgtggt cgggatgtga agtttactct tgaagtgctc cggggtgata
     6421 gtgttgagaa aacctctcgg gtatggtcag gtaatgaacg tgaccaggag ctgcttactg
     6481 aggacgcact ggatgatctc atcccttctt ttctactgac tggtcaacag acaccggcgt
     6541 tcggtcgaag agtatctggt gtcatagaaa ttgccgatgg gagtcgccgt cgtaaagctg
     6601 ctgcacttac cgaaagtgat tatcgtgttc tggttggcga gctggatgat gagcagatgg
     6661 ctgcattatc cagattgggt aacgattatc gcccaacaag tgcttatgaa cgtggtcagc
     6721 gttatgcaag ccgattgcag aatgaatttg ctggaaatat ttctgcgctg gctgatgcgg
     6781 aaaatatttc acgtaagatt attacccgct gtatcaacac cgccaaattg cctaaatcag
     6841 ttgttgctct tttttctcac cccggtgaac tatctgcccg gtcaggtgat gcacttcaaa
     6901 aagcctttac agataaagag gaattactta agcagcaggc atctaacctt catgagcaga
     6961 aaaaagctgg ggtgatattt gaagctgaag aagttatcac tcttttaact tctgtgctta
     7021 aaacgtcatc tgcatcaaga actagtttaa gctcacgaca tcagtttgct cctggagcga
     7081 cagtattgta taagggcgat aaaatggtgc ttaacctgga caggtcacgt gttccaactg
     7141 agtgtataga gaaaattgag gccattctta aggaacttga aaagccagca ccctgatgcg
     7201 accacgtttt agtctacgtt tatctgtctt tacttaatgt cctttgttac aggccagaaa
     7261 gcataactgg cctgaatatt ctctctgggc ccactgttcc acttgtatcg tcggtctgat
     7321 aatcagactg ggaccacggt cccactcgta tcgtcggtct gattattagt ctgggaccac
     7381 ggtcccactc gtatcgtcgg tctgattatt agtctgggac cacggtccca ctcgtatcgt
     7441 cggtctgata atcagactgg gaccacggtc ccactcgtat cgtcggtctg attattagtc
     7501 tgggaccatg gtcccactcg tatcgtcggt ctgattatta gtctgggacc acggtcccac
     7561 tcgtatcgtc ggtctgatta ttagtctgga accacggtcc cactcgtatc gtcggtctga
     7621 ttattagtct gggaccacgg tcccactcgt atcgtcggtc tgattattag tctgggacca
     7681 cgatcccact cgtgttgtcg gtctgattat cggtctggga ccacggtccc acttgtattg
     7741 tcgatcagac tatcagcgtg agactacgat tccatcaatg cctgtcaagg gcaagtattg
     7801 acatgtcgtc gtaacctgta gaacggagta acctcggtgt gcggttgtat gcctgctgtg
     7861 gattgctgct gtgtcctgct tatccacaac attttgcgca cggttatgtg gacaaaatac
     7921 ctggttaccc aggccgtgcc ggcacgttaa ccgggctgca tccgatgcaa gtgtgtcgct
     7981 gtcgacgcaa gcggccgca
//
