LOCUS       sequence-465761-        8025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMOBK360_VR: Vector Right (VR) 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 8025)
  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 8025)
  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: POC1520
FEATURES             Location/Qualifiers
     source          1..8025
                     /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        1718..1822
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1823..2617
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3432..3651
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3742..4497
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4500..4750
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5076..6251
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6251..7222
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7295..7768
                     /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 agactgagac caagtcgtga
     1681 gaggtactgc accacgaagt gtgcggccgc cttcgtgcgc ggaaccccta tttgtttatt
     1741 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1801 ataatattga aaaaggaaga gtatggctaa aatgagaata tcaccggaat tgaaaaaact
     1861 gatcgaaaaa taccgctgcg taaaagatac ggaaggaatg tctcctgcta aggtatataa
     1921 gctggtggga gaaaatgaaa acctatattt aaaaatgacg gacagccggt ataaagggac
     1981 cacctatgat gtggaacggg aaaaggacat gatgctatgg ctggaaggaa agctgcctgt
     2041 tccaaaggtc ctgcactttg aacggcatga tggctggagc aatctgctca tgagtgaggc
     2101 cgatggcgtc ctttgctcgg aagagtatga agatgaacaa agccctgaaa agattatcga
     2161 gctgtatgcg gagtgcatca ggctctttca ctccatcgac atatcggatt gtccctatac
     2221 gaatagctta gacagccgct tagccgaatt ggattactta ctgaataacg atctggccga
     2281 tgtggattgc gaaaactggg aagaggacac tccatttaaa gatccgcgcg agctgtatga
     2341 ttttttaaag acggaaaagc ccgaagagga acttgtcttt tcccacggcg acctgggaga
     2401 cagcaacatc tttgtgaaag atggcaaagt aagtggcttt attgatcttg ggagaagcgg
     2461 cagggcggac aagtggtatg acattgcctt ctgcgtccgg tcgctcaggg aggatatcgg
     2521 ggaagaacag tatgtcgagc tattttttga cttactgggg atcaagcctg attgggagaa
     2581 aataaaatat tatattttac tggatgaatt gttttaggag ctggagcggc gtaaccgtcg
     2641 cacaggaagg acagagaaag cgcggatctg ggaagtgacg gacagaacgg tcaggacctg
     2701 gattggggag gcggttgccg ccgctgctgc tgacggtgtg acgttctctg ttccggtcac
     2761 accacatacg ttccgccatt cctatgcgat gcacatgctg tatgccggta taccgctgaa
     2821 agttctgcaa agcctgatgg gacataagtc catcagttca acggaagtct acacgaaggt
     2881 ttttgcgctg gatgtggctg cccggctccg ggtgcagttt gcgatgccgg agtctgatgc
     2941 ggttgcgatg ctgaaacaat tatcctgaga ataaatgcct tggcctttat atggaaatgt
     3001 ggaactgagt ggatatgctg tttttgtctg ttaaacagag aagctggctg ttatccactg
     3061 agaagcgaac gaaacagtcg ggaaaatctc ccattatcgt agagatccgc attattaatc
     3121 tcaggagcct gtgtagcgtt tataggaagt agtgttctgt catgatgcct gcaagcggta
     3181 acgaaaacga tttgaatatg ccttcaggaa caatagaaat cttcgtgcgg tgttacgttg
     3241 aagtggagcg gattatgtca gcaatggaca gaacaaccta atgaacacag aaccatgatg
     3301 tggtctgtcc ttttacagcc agtagtgctc gccgcagtcg agcgacaggg cgaagccctc
     3361 gagtgagcga ggaagcacca gggaacagca cttatatatt ctgcttacac acgatgcctg
     3421 aaaaaacttc ccttggggtt atccacttat ccacggggat atttttataa ttattttttt
     3481 tatagttttt agatcttctt ttttagagcg ccttgtaggc ctttatccat gctggttcta
     3541 gagaaggtgt tgtgacaaat tgccctttca gtgtgacaaa tcaccctcaa atgacagtcc
     3601 tgtctgtgac aaattgccct taaccctgtg acaaattgcc ctcagaagaa gctgtttttt
     3661 cacaaagtta tccctgctta ttgactcttt tttatttagt gtgacaatct aaaaacttgt
     3721 cacacttcac atggatctgt catggcggaa acagcggtta tcaatcacaa gaaacgtaaa
     3781 aatagcccgc gaatcgtcca gtcaaacgac ctcactgagg cggcatatag tctctcccgg
     3841 gatcaaaaac gtatgctgta tctgttcgtt gaccagatca gaaaatctga tggcacccta
     3901 caggaacatg acggtatctg cgagatccat gttgctaaat atgctgaaat attcggattg
     3961 acctctgcgg aagccagtaa ggatatacgg caggcattga agagtttcgc ggggaaggaa
     4021 gtggtttttt atcgccctga agaggatgcc ggcgatgaaa aaggctatga atcttttcct
     4081 tggtttatca aacgtgcgca cagtccatcc agagggcttt acagtgtaca tatcaaccca
     4141 tatctcattc ccttctttat cgggttacag aaccggttta cgcagtttcg gcttagtgaa
     4201 acaaaagaaa tcaccaatcc gtatgccatg cgtttatacg aatccctgtg tcagtatcgt
     4261 aagccggatg gctcaggcat cgtgtctctg aaaatcgact ggatcataga gcgttaccag
     4321 ctgcctcaaa gttaccagcg tatgcctgac ttccgccgcc gcttcctgca ggtctgtgtt
     4381 aatgagatca acagcagaac tccaatgcgc ctctcataca ttgagaaaaa gaaaggccgc
     4441 cagacgactc atatcgtatt ttccttccgc gatatcactt ccatgacgac aggatagtct
     4501 gagggttatc tgtcacagat ttgagggtgg ttcgtcacat ttgttctgac ctactgaggg
     4561 taatttgtca cagttttgct gtttccttca gcctgcatgg attttctcat actttttgaa
     4621 ctgtaatttt taaggaagcc aaatttgagg gcagtttgtc acagttgatt tccttctctt
     4681 tcccttcgtc atgtgacctg atatcggggg ttagttcgtc atcattgatg agggttgatt
     4741 atcacagttt attactctga attggctatc cgcgtgtgta cctctacctg gagtttttcc
     4801 cacggtggat atttcttctt gcgctgagcg taagagctat ctgacagaac agttcttctt
     4861 tgcttcctcg ccagttcgct cgctatgctc ggttacacgg ctgcggcgag cgctagtgat
     4921 aataagtgac tgaggtatgt ggtgttctta tctccttttg tagtgttgct cttattttaa
     4981 acaactttgc ggttttttga tgactttgcg attttgttgt tgctttgcag taaattgcaa
     5041 gatttaataa aaaaacgcaa agcaatgatt aaaggatgtt cagaatgaaa ctcatggaaa
     5101 cacttaacca gtgcataaac gctggtcatg aaatgacgaa ggctatcgcc attgcacagt
     5161 ttaatgatga cagcccggaa gcgaggaaaa taacccggcg ctggagaata ggtgaagcag
     5221 cggatttagt tggggtttct tctcaggcta tcagagatgc cgagaaagca gggcgactac
     5281 cgcacccgga tatggaaatt cgaggacggg ttgagcaacg tgttggttat acaattgaac
     5341 aaattaatca tatgcgtgat gtgtttggta cgcgattgcg acgtgctgag gacgtatttc
     5401 caccggtgat cggggttgct gcccataaag gtggcgttta caaaacctca gtttctgttc
     5461 atcttgctca ggatctggct ctgaaggggc tacgtgtttt gctcgtggaa ggtaacgacc
     5521 cccagggaac agcctcaatg tatcacggat gggtaccaga tcttcatatt catgcagaag
     5581 atactctcct gcctttctat cttggggaaa aggacgatgt cacttatgca ataaagccca
     5641 cttgctggcc ggggcttgac attattcctt cctgtctggc tctgcaccgt attgaaactg
     5701 agttaatggg caaatttgat gaaggtaaac tgcccaccga tccacacctg atgctccgac
     5761 tggccattga aactgttgct catgactatg atgtcatagt tattgacagc gcgcctaacc
     5821 tgggtatcgg cacgattaat gtcgtatgtg ctgctgatgt gctgattgtt cccacgcctg
     5881 ctgagttgtt tgactacacc tccgcactgc agtttttcga tatgcttcgt gatctgctca
     5941 agaacgttga tcttaaaggg ttcgagcctg atgtacgtat tttgcttacc aaatacagca
     6001 atagtaatgg ctctcagtcc ccgtggatgg aggagcaaat tcgggatgcc tggggaagca
     6061 tggttctaaa aaatgttgta cgtgaaacgg atgaagttgg taaaggtcag atccggatga
     6121 gaactgtttt tgaacaggcc attgatcaac gcagttcaac tggtgcctgg agaaatgctc
     6181 tttctatttg ggaacctgtc tgcaatgaaa ttttcgatcg tctgattaaa ccacgctggg
     6241 agattagata atgaagcgtg cgcctgttat tccaaaacat acgctcaata ctcaaccggt
     6301 tgaagatact tcgttatcga caccagctgc cccgatggtg gattcgttaa ttgcgcgcgt
     6361 aggagtaatg gctcgcggta atgccattac tttgcctgta tgtggtcggg atgtgaagtt
     6421 tactcttgaa gtgctccggg gtgatagtgt tgagaaaacc tctcgggtat ggtcaggtaa
     6481 tgaacgtgac caggagctgc ttactgagga cgcactggat gatctcatcc cttcttttct
     6541 actgactggt caacagacac cggcgttcgg tcgaagagta tctggtgtca tagaaattgc
     6601 cgatgggagt cgccgtcgta aagctgctgc acttaccgaa agtgattatc gtgttctggt
     6661 tggcgagctg gatgatgagc agatggctgc attatccaga ttgggtaacg attatcgccc
     6721 aacaagtgct tatgaacgtg gtcagcgtta tgcaagccga ttgcagaatg aatttgctgg
     6781 aaatatttct gcgctggctg atgcggaaaa tatttcacgt aagattatta cccgctgtat
     6841 caacaccgcc aaattgccta aatcagttgt tgctcttttt tctcaccccg gtgaactatc
     6901 tgcccggtca ggtgatgcac ttcaaaaagc ctttacagat aaagaggaat tacttaagca
     6961 gcaggcatct aaccttcatg agcagaaaaa agctggggtg atatttgaag ctgaagaagt
     7021 tatcactctt ttaacttctg tgcttaaaac gtcatctgca tcaagaacta gtttaagctc
     7081 acgacatcag tttgctcctg gagcgacagt attgtataag ggcgataaaa tggtgcttaa
     7141 cctggacagg tcacgtgttc caactgagtg tatagagaaa attgaggcca ttcttaagga
     7201 acttgaaaag ccagcaccct gatgcgacca cgttttagtc tacgtttatc tgtctttact
     7261 taatgtcctt tgttacaggc cagaaagcat aactggcctg aatattctct ctgggcccac
     7321 tgttccactt gtatcgtcgg tctgataatc agactgggac cacggtccca ctcgtatcgt
     7381 cggtctgatt attagtctgg gaccacggtc ccactcgtat cgtcggtctg attattagtc
     7441 tgggaccacg gtcccactcg tatcgtcggt ctgataatca gactgggacc acggtcccac
     7501 tcgtatcgtc ggtctgatta ttagtctggg accatggtcc cactcgtatc gtcggtctga
     7561 ttattagtct gggaccacgg tcccactcgt atcgtcggtc tgattattag tctggaacca
     7621 cggtcccact cgtatcgtcg gtctgattat tagtctggga ccacggtccc actcgtatcg
     7681 tcggtctgat tattagtctg ggaccacgat cccactcgtg ttgtcggtct gattatcggt
     7741 ctgggaccac ggtcccactt gtattgtcga tcagactatc agcgtgagac tacgattcca
     7801 tcaatgcctg tcaagggcaa gtattgacat gtcgtcgtaa cctgtagaac ggagtaacct
     7861 cggtgtgcgg ttgtatgcct gctgtggatt gctgctgtgt cctgcttatc cacaacattt
     7921 tgcgcacggt tatgtggaca aaatacctgg ttacccaggc cgtgccggca cgttaaccgg
     7981 gctgcatccg atgcaagtgt gtcgctgtcg acgcaagcgg ccgca
//
