LOCUS       sequence-465768-        8019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMOBK360_VM: Vector Middle (VM) of the set (Kanamycin R) to be 
            methylated by the M.Osp807II BsaI-associated switch methylase. Used 
            for the assembly using the methylation protection approach.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8019)
  AUTHORS   Flores-Fernandez CN, Lin D, Robins K, O'Callaghan CA
  TITLE     UniClo: scarless hierarchical DNA assembly without sequence 
            constraint.
  JOURNAL   Nucleic Acids Res. 2025 Jun 20;53(12):gkaf548. doi: 
            10.1093/nar/gkaf548.
  PUBMED    40548934
REFERENCE   2  (bases 1 to 8019)
  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."; date: "2025-06-20"; volume: "53(12):gkaf548. doi"; 
            pages: " 10.1093/nar/gkaf548"
COMMENT     Sequence Label: POC1519
FEATURES             Location/Qualifiers
     source          1..8019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      233..821
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     722..741
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     975..992
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1109..1130
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1145..1150,1151..1168,1169..1175)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1183..1199
                     /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     1188..1210
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1207..1223
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1207..1223
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1219..1542
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1236..1292
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1293..1310)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1293..1309)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1302..1324)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1518..1537)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1712..1816
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1817..2611
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3426..3645
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3736..4491
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4494..4744
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5070..6245
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6245..7216
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7289..7762
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
ORIGIN
        1 cactctgtgg tgcagtacct ctcacgactt ggtctcctga gaccggccta gcaggtgtga
       61 gtttgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
      121 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
      181 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
      241 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      301 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      361 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
      421 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      481 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      541 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      601 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      661 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
      721 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
      781 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
      841 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
      901 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
      961 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga tgagcgccca atacgcaaac
     1021 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     1081 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     1141 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     1201 ttcacacagg aaacagctat gaccatgatt acgccaagct tgcatgcctg caggtcgact
     1261 ctagaggatc cccgggtacc gagctcgaat tcactggccg tcgttttaca acgtcgtgac
     1321 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     1381 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     1441 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     1501 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     1561 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     1621 caagctgtga caagtaccac ctggaacgcc aaaggtctca agaccaagtc gtgagaggta
     1681 ctgcaccacg aagtgtgcgg ccgccttcgt gcgcggaacc cctatttgtt tatttttcta
     1741 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1801 ttgaaaaagg aagagtatgg ctaaaatgag aatatcaccg gaattgaaaa aactgatcga
     1861 aaaataccgc tgcgtaaaag atacggaagg aatgtctcct gctaaggtat ataagctggt
     1921 gggagaaaat gaaaacctat atttaaaaat gacggacagc cggtataaag ggaccaccta
     1981 tgatgtggaa cgggaaaagg acatgatgct atggctggaa ggaaagctgc ctgttccaaa
     2041 ggtcctgcac tttgaacggc atgatggctg gagcaatctg ctcatgagtg aggccgatgg
     2101 cgtcctttgc tcggaagagt atgaagatga acaaagccct gaaaagatta tcgagctgta
     2161 tgcggagtgc atcaggctct ttcactccat cgacatatcg gattgtccct atacgaatag
     2221 cttagacagc cgcttagccg aattggatta cttactgaat aacgatctgg ccgatgtgga
     2281 ttgcgaaaac tgggaagagg acactccatt taaagatccg cgcgagctgt atgatttttt
     2341 aaagacggaa aagcccgaag aggaacttgt cttttcccac ggcgacctgg gagacagcaa
     2401 catctttgtg aaagatggca aagtaagtgg ctttattgat cttgggagaa gcggcagggc
     2461 ggacaagtgg tatgacattg ccttctgcgt ccggtcgctc agggaggata tcggggaaga
     2521 acagtatgtc gagctatttt ttgacttact ggggatcaag cctgattggg agaaaataaa
     2581 atattatatt ttactggatg aattgtttta ggagctggag cggcgtaacc gtcgcacagg
     2641 aaggacagag aaagcgcgga tctgggaagt gacggacaga acggtcagga cctggattgg
     2701 ggaggcggtt gccgccgctg ctgctgacgg tgtgacgttc tctgttccgg tcacaccaca
     2761 tacgttccgc cattcctatg cgatgcacat gctgtatgcc ggtataccgc tgaaagttct
     2821 gcaaagcctg atgggacata agtccatcag ttcaacggaa gtctacacga aggtttttgc
     2881 gctggatgtg gctgcccggc tccgggtgca gtttgcgatg ccggagtctg atgcggttgc
     2941 gatgctgaaa caattatcct gagaataaat gccttggcct ttatatggaa atgtggaact
     3001 gagtggatat gctgtttttg tctgttaaac agagaagctg gctgttatcc actgagaagc
     3061 gaacgaaaca gtcgggaaaa tctcccatta tcgtagagat ccgcattatt aatctcagga
     3121 gcctgtgtag cgtttatagg aagtagtgtt ctgtcatgat gcctgcaagc ggtaacgaaa
     3181 acgatttgaa tatgccttca ggaacaatag aaatcttcgt gcggtgttac gttgaagtgg
     3241 agcggattat gtcagcaatg gacagaacaa cctaatgaac acagaaccat gatgtggtct
     3301 gtccttttac agccagtagt gctcgccgca gtcgagcgac agggcgaagc cctcgagtga
     3361 gcgaggaagc accagggaac agcacttata tattctgctt acacacgatg cctgaaaaaa
     3421 cttcccttgg ggttatccac ttatccacgg ggatattttt ataattattt tttttatagt
     3481 ttttagatct tcttttttag agcgccttgt aggcctttat ccatgctggt tctagagaag
     3541 gtgttgtgac aaattgccct ttcagtgtga caaatcaccc tcaaatgaca gtcctgtctg
     3601 tgacaaattg cccttaaccc tgtgacaaat tgccctcaga agaagctgtt ttttcacaaa
     3661 gttatccctg cttattgact cttttttatt tagtgtgaca atctaaaaac ttgtcacact
     3721 tcacatggat ctgtcatggc ggaaacagcg gttatcaatc acaagaaacg taaaaatagc
     3781 ccgcgaatcg tccagtcaaa cgacctcact gaggcggcat atagtctctc ccgggatcaa
     3841 aaacgtatgc tgtatctgtt cgttgaccag atcagaaaat ctgatggcac cctacaggaa
     3901 catgacggta tctgcgagat ccatgttgct aaatatgctg aaatattcgg attgacctct
     3961 gcggaagcca gtaaggatat acggcaggca ttgaagagtt tcgcggggaa ggaagtggtt
     4021 ttttatcgcc ctgaagagga tgccggcgat gaaaaaggct atgaatcttt tccttggttt
     4081 atcaaacgtg cgcacagtcc atccagaggg ctttacagtg tacatatcaa cccatatctc
     4141 attcccttct ttatcgggtt acagaaccgg tttacgcagt ttcggcttag tgaaacaaaa
     4201 gaaatcacca atccgtatgc catgcgttta tacgaatccc tgtgtcagta tcgtaagccg
     4261 gatggctcag gcatcgtgtc tctgaaaatc gactggatca tagagcgtta ccagctgcct
     4321 caaagttacc agcgtatgcc tgacttccgc cgccgcttcc tgcaggtctg tgttaatgag
     4381 atcaacagca gaactccaat gcgcctctca tacattgaga aaaagaaagg ccgccagacg
     4441 actcatatcg tattttcctt ccgcgatatc acttccatga cgacaggata gtctgagggt
     4501 tatctgtcac agatttgagg gtggttcgtc acatttgttc tgacctactg agggtaattt
     4561 gtcacagttt tgctgtttcc ttcagcctgc atggattttc tcatactttt tgaactgtaa
     4621 tttttaagga agccaaattt gagggcagtt tgtcacagtt gatttccttc tctttccctt
     4681 cgtcatgtga cctgatatcg ggggttagtt cgtcatcatt gatgagggtt gattatcaca
     4741 gtttattact ctgaattggc tatccgcgtg tgtacctcta cctggagttt ttcccacggt
     4801 ggatatttct tcttgcgctg agcgtaagag ctatctgaca gaacagttct tctttgcttc
     4861 ctcgccagtt cgctcgctat gctcggttac acggctgcgg cgagcgctag tgataataag
     4921 tgactgaggt atgtggtgtt cttatctcct tttgtagtgt tgctcttatt ttaaacaact
     4981 ttgcggtttt ttgatgactt tgcgattttg ttgttgcttt gcagtaaatt gcaagattta
     5041 ataaaaaaac gcaaagcaat gattaaagga tgttcagaat gaaactcatg gaaacactta
     5101 accagtgcat aaacgctggt catgaaatga cgaaggctat cgccattgca cagtttaatg
     5161 atgacagccc ggaagcgagg aaaataaccc ggcgctggag aataggtgaa gcagcggatt
     5221 tagttggggt ttcttctcag gctatcagag atgccgagaa agcagggcga ctaccgcacc
     5281 cggatatgga aattcgagga cgggttgagc aacgtgttgg ttatacaatt gaacaaatta
     5341 atcatatgcg tgatgtgttt ggtacgcgat tgcgacgtgc tgaggacgta tttccaccgg
     5401 tgatcggggt tgctgcccat aaaggtggcg tttacaaaac ctcagtttct gttcatcttg
     5461 ctcaggatct ggctctgaag gggctacgtg ttttgctcgt ggaaggtaac gacccccagg
     5521 gaacagcctc aatgtatcac ggatgggtac cagatcttca tattcatgca gaagatactc
     5581 tcctgccttt ctatcttggg gaaaaggacg atgtcactta tgcaataaag cccacttgct
     5641 ggccggggct tgacattatt ccttcctgtc tggctctgca ccgtattgaa actgagttaa
     5701 tgggcaaatt tgatgaaggt aaactgccca ccgatccaca cctgatgctc cgactggcca
     5761 ttgaaactgt tgctcatgac tatgatgtca tagttattga cagcgcgcct aacctgggta
     5821 tcggcacgat taatgtcgta tgtgctgctg atgtgctgat tgttcccacg cctgctgagt
     5881 tgtttgacta cacctccgca ctgcagtttt tcgatatgct tcgtgatctg ctcaagaacg
     5941 ttgatcttaa agggttcgag cctgatgtac gtattttgct taccaaatac agcaatagta
     6001 atggctctca gtccccgtgg atggaggagc aaattcggga tgcctgggga agcatggttc
     6061 taaaaaatgt tgtacgtgaa acggatgaag ttggtaaagg tcagatccgg atgagaactg
     6121 tttttgaaca ggccattgat caacgcagtt caactggtgc ctggagaaat gctctttcta
     6181 tttgggaacc tgtctgcaat gaaattttcg atcgtctgat taaaccacgc tgggagatta
     6241 gataatgaag cgtgcgcctg ttattccaaa acatacgctc aatactcaac cggttgaaga
     6301 tacttcgtta tcgacaccag ctgccccgat ggtggattcg ttaattgcgc gcgtaggagt
     6361 aatggctcgc ggtaatgcca ttactttgcc tgtatgtggt cgggatgtga agtttactct
     6421 tgaagtgctc cggggtgata gtgttgagaa aacctctcgg gtatggtcag gtaatgaacg
     6481 tgaccaggag ctgcttactg aggacgcact ggatgatctc atcccttctt ttctactgac
     6541 tggtcaacag acaccggcgt tcggtcgaag agtatctggt gtcatagaaa ttgccgatgg
     6601 gagtcgccgt cgtaaagctg ctgcacttac cgaaagtgat tatcgtgttc tggttggcga
     6661 gctggatgat gagcagatgg ctgcattatc cagattgggt aacgattatc gcccaacaag
     6721 tgcttatgaa cgtggtcagc gttatgcaag ccgattgcag aatgaatttg ctggaaatat
     6781 ttctgcgctg gctgatgcgg aaaatatttc acgtaagatt attacccgct gtatcaacac
     6841 cgccaaattg cctaaatcag ttgttgctct tttttctcac cccggtgaac tatctgcccg
     6901 gtcaggtgat gcacttcaaa aagcctttac agataaagag gaattactta agcagcaggc
     6961 atctaacctt catgagcaga aaaaagctgg ggtgatattt gaagctgaag aagttatcac
     7021 tcttttaact tctgtgctta aaacgtcatc tgcatcaaga actagtttaa gctcacgaca
     7081 tcagtttgct cctggagcga cagtattgta taagggcgat aaaatggtgc ttaacctgga
     7141 caggtcacgt gttccaactg agtgtataga gaaaattgag gccattctta aggaacttga
     7201 aaagccagca ccctgatgcg accacgtttt agtctacgtt tatctgtctt tacttaatgt
     7261 cctttgttac aggccagaaa gcataactgg cctgaatatt ctctctgggc ccactgttcc
     7321 acttgtatcg tcggtctgat aatcagactg ggaccacggt cccactcgta tcgtcggtct
     7381 gattattagt ctgggaccac ggtcccactc gtatcgtcgg tctgattatt agtctgggac
     7441 cacggtccca ctcgtatcgt cggtctgata atcagactgg gaccacggtc ccactcgtat
     7501 cgtcggtctg attattagtc tgggaccatg gtcccactcg tatcgtcggt ctgattatta
     7561 gtctgggacc acggtcccac tcgtatcgtc ggtctgatta ttagtctgga accacggtcc
     7621 cactcgtatc gtcggtctga ttattagtct gggaccacgg tcccactcgt atcgtcggtc
     7681 tgattattag tctgggacca cgatcccact cgtgttgtcg gtctgattat cggtctggga
     7741 ccacggtccc acttgtattg tcgatcagac tatcagcgtg agactacgat tccatcaatg
     7801 cctgtcaagg gcaagtattg acatgtcgtc gtaacctgta gaacggagta acctcggtgt
     7861 gcggttgtat gcctgctgtg gattgctgct gtgtcctgct tatccacaac attttgcgca
     7921 cggttatgtg gacaaaatac ctggttaccc aggccgtgcc ggcacgttaa ccgggctgca
     7981 tccgatgcaa gtgtgtcgct gtcgacgcaa gcggccgca
//
