LOCUS       sequence-465742-        7994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMOBC360_VM: Vector Middle (VM) of the set (Chloramphenicol 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 7994)
  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 7994)
  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: POC1525
FEATURES             Location/Qualifiers
     source          1..7994
                     /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        1824..1926
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1927..2586
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1993..2012)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      3401..3620
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3711..4466
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4469..4719
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5045..6220
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6220..7191
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7264..7737
                     /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 ccgccttcag cggccgcacg aataaatacc tgtgacggaa
     1741 gatcacttcg cagaataaat aaatcctggt gtccctgttg ataccgggaa gccctgggcc
     1801 aacttttggc gaaaatgaga cgttgatcgg cacgtaagag gttccaactt tcaccataat
     1861 gaaataagat cactaccggg cgtatttttt gagttatcga gattttcagg agctaaggaa
     1921 gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt
     1981 aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag
     2041 ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc
     2101 tttattcaca ttcttgcccg cctgatgaat gctcatccgg agttccgtat ggcaatgaaa
     2161 gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa
     2221 actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac
     2281 atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt
     2341 attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta
     2401 aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg
     2461 caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc
     2521 ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg
     2581 gcgtaagagc tggagcggcg taaccgtcgc acaggaagga cagagaaagc gcggatctgg
     2641 gaagtgacgg acagaacggt caggacctgg attggggagg cggttgccgc cgctgctgct
     2701 gacggtgtga cgttctctgt tccggtcaca ccacatacgt tccgccattc ctatgcgatg
     2761 cacatgctgt atgccggtat accgctgaaa gttctgcaaa gcctgatggg acataagtcc
     2821 atcagttcaa cggaagtcta cacgaaggtt tttgcgctgg atgtggctgc ccggctccgg
     2881 gtgcagtttg cgatgccgga gtctgatgcg gttgcgatgc tgaaacaatt atcctgagaa
     2941 taaatgcctt ggcctttata tggaaatgtg gaactgagtg gatatgctgt ttttgtctgt
     3001 taaacagaga agctggctgt tatccactga gaagcgaacg aaacagtcgg gaaaatctcc
     3061 cattatcgta gagatccgca ttattaatct caggagcctg tgtagcgttt ataggaagta
     3121 gtgttctgtc atgatgcctg caagcggtaa cgaaaacgat ttgaatatgc cttcaggaac
     3181 aatagaaatc ttcgtgcggt gttacgttga agtggagcgg attatgtcag caatggacag
     3241 aacaacctaa tgaacacaga accatgatgt ggtctgtcct tttacagcca gtagtgctcg
     3301 ccgcagtcga gcgacagggc gaagccctcg agtgagcgag gaagcaccag ggaacagcac
     3361 ttatatattc tgcttacaca cgatgcctga aaaaacttcc cttggggtta tccacttatc
     3421 cacggggata tttttataat tatttttttt atagttttta gatcttcttt tttagagcgc
     3481 cttgtaggcc tttatccatg ctggttctag agaaggtgtt gtgacaaatt gccctttcag
     3541 tgtgacaaat caccctcaaa tgacagtcct gtctgtgaca aattgccctt aaccctgtga
     3601 caaattgccc tcagaagaag ctgttttttc acaaagttat ccctgcttat tgactctttt
     3661 ttatttagtg tgacaatcta aaaacttgtc acacttcaca tggatctgtc atggcggaaa
     3721 cagcggttat caatcacaag aaacgtaaaa atagcccgcg aatcgtccag tcaaacgacc
     3781 tcactgaggc ggcatatagt ctctcccggg atcaaaaacg tatgctgtat ctgttcgttg
     3841 accagatcag aaaatctgat ggcaccctac aggaacatga cggtatctgc gagatccatg
     3901 ttgctaaata tgctgaaata ttcggattga cctctgcgga agccagtaag gatatacggc
     3961 aggcattgaa gagtttcgcg gggaaggaag tggtttttta tcgccctgaa gaggatgccg
     4021 gcgatgaaaa aggctatgaa tcttttcctt ggtttatcaa acgtgcgcac agtccatcca
     4081 gagggcttta cagtgtacat atcaacccat atctcattcc cttctttatc gggttacaga
     4141 accggtttac gcagtttcgg cttagtgaaa caaaagaaat caccaatccg tatgccatgc
     4201 gtttatacga atccctgtgt cagtatcgta agccggatgg ctcaggcatc gtgtctctga
     4261 aaatcgactg gatcatagag cgttaccagc tgcctcaaag ttaccagcgt atgcctgact
     4321 tccgccgccg cttcctgcag gtctgtgtta atgagatcaa cagcagaact ccaatgcgcc
     4381 tctcatacat tgagaaaaag aaaggccgcc agacgactca tatcgtattt tccttccgcg
     4441 atatcacttc catgacgaca ggatagtctg agggttatct gtcacagatt tgagggtggt
     4501 tcgtcacatt tgttctgacc tactgagggt aatttgtcac agttttgctg tttccttcag
     4561 cctgcatgga ttttctcata ctttttgaac tgtaattttt aaggaagcca aatttgaggg
     4621 cagtttgtca cagttgattt ccttctcttt cccttcgtca tgtgacctga tatcgggggt
     4681 tagttcgtca tcattgatga gggttgatta tcacagttta ttactctgaa ttggctatcc
     4741 gcgtgtgtac ctctacctgg agtttttccc acggtggata tttcttcttg cgctgagcgt
     4801 aagagctatc tgacagaaca gttcttcttt gcttcctcgc cagttcgctc gctatgctcg
     4861 gttacacggc tgcggcgagc gctagtgata ataagtgact gaggtatgtg gtgttcttat
     4921 ctccttttgt agtgttgctc ttattttaaa caactttgcg gttttttgat gactttgcga
     4981 ttttgttgtt gctttgcagt aaattgcaag atttaataaa aaaacgcaaa gcaatgatta
     5041 aaggatgttc agaatgaaac tcatggaaac acttaaccag tgcataaacg ctggtcatga
     5101 aatgacgaag gctatcgcca ttgcacagtt taatgatgac agcccggaag cgaggaaaat
     5161 aacccggcgc tggagaatag gtgaagcagc ggatttagtt ggggtttctt ctcaggctat
     5221 cagagatgcc gagaaagcag ggcgactacc gcacccggat atggaaattc gaggacgggt
     5281 tgagcaacgt gttggttata caattgaaca aattaatcat atgcgtgatg tgtttggtac
     5341 gcgattgcga cgtgctgagg acgtatttcc accggtgatc ggggttgctg cccataaagg
     5401 tggcgtttac aaaacctcag tttctgttca tcttgctcag gatctggctc tgaaggggct
     5461 acgtgttttg ctcgtggaag gtaacgaccc ccagggaaca gcctcaatgt atcacggatg
     5521 ggtaccagat cttcatattc atgcagaaga tactctcctg cctttctatc ttggggaaaa
     5581 ggacgatgtc acttatgcaa taaagcccac ttgctggccg gggcttgaca ttattccttc
     5641 ctgtctggct ctgcaccgta ttgaaactga gttaatgggc aaatttgatg aaggtaaact
     5701 gcccaccgat ccacacctga tgctccgact ggccattgaa actgttgctc atgactatga
     5761 tgtcatagtt attgacagcg cgcctaacct gggtatcggc acgattaatg tcgtatgtgc
     5821 tgctgatgtg ctgattgttc ccacgcctgc tgagttgttt gactacacct ccgcactgca
     5881 gtttttcgat atgcttcgtg atctgctcaa gaacgttgat cttaaagggt tcgagcctga
     5941 tgtacgtatt ttgcttacca aatacagcaa tagtaatggc tctcagtccc cgtggatgga
     6001 ggagcaaatt cgggatgcct ggggaagcat ggttctaaaa aatgttgtac gtgaaacgga
     6061 tgaagttggt aaaggtcaga tccggatgag aactgttttt gaacaggcca ttgatcaacg
     6121 cagttcaact ggtgcctgga gaaatgctct ttctatttgg gaacctgtct gcaatgaaat
     6181 tttcgatcgt ctgattaaac cacgctggga gattagataa tgaagcgtgc gcctgttatt
     6241 ccaaaacata cgctcaatac tcaaccggtt gaagatactt cgttatcgac accagctgcc
     6301 ccgatggtgg attcgttaat tgcgcgcgta ggagtaatgg ctcgcggtaa tgccattact
     6361 ttgcctgtat gtggtcggga tgtgaagttt actcttgaag tgctccgggg tgatagtgtt
     6421 gagaaaacct ctcgggtatg gtcaggtaat gaacgtgacc aggagctgct tactgaggac
     6481 gcactggatg atctcatccc ttcttttcta ctgactggtc aacagacacc ggcgttcggt
     6541 cgaagagtat ctggtgtcat agaaattgcc gatgggagtc gccgtcgtaa agctgctgca
     6601 cttaccgaaa gtgattatcg tgttctggtt ggcgagctgg atgatgagca gatggctgca
     6661 ttatccagat tgggtaacga ttatcgccca acaagtgctt atgaacgtgg tcagcgttat
     6721 gcaagccgat tgcagaatga atttgctgga aatatttctg cgctggctga tgcggaaaat
     6781 atttcacgta agattattac ccgctgtatc aacaccgcca aattgcctaa atcagttgtt
     6841 gctctttttt ctcaccccgg tgaactatct gcccggtcag gtgatgcact tcaaaaagcc
     6901 tttacagata aagaggaatt acttaagcag caggcatcta accttcatga gcagaaaaaa
     6961 gctggggtga tatttgaagc tgaagaagtt atcactcttt taacttctgt gcttaaaacg
     7021 tcatctgcat caagaactag tttaagctca cgacatcagt ttgctcctgg agcgacagta
     7081 ttgtataagg gcgataaaat ggtgcttaac ctggacaggt cacgtgttcc aactgagtgt
     7141 atagagaaaa ttgaggccat tcttaaggaa cttgaaaagc cagcaccctg atgcgaccac
     7201 gttttagtct acgtttatct gtctttactt aatgtccttt gttacaggcc agaaagcata
     7261 actggcctga atattctctc tgggcccact gttccacttg tatcgtcggt ctgataatca
     7321 gactgggacc acggtcccac tcgtatcgtc ggtctgatta ttagtctggg accacggtcc
     7381 cactcgtatc gtcggtctga ttattagtct gggaccacgg tcccactcgt atcgtcggtc
     7441 tgataatcag actgggacca cggtcccact cgtatcgtcg gtctgattat tagtctggga
     7501 ccatggtccc actcgtatcg tcggtctgat tattagtctg ggaccacggt cccactcgta
     7561 tcgtcggtct gattattagt ctggaaccac ggtcccactc gtatcgtcgg tctgattatt
     7621 agtctgggac cacggtccca ctcgtatcgt cggtctgatt attagtctgg gaccacgatc
     7681 ccactcgtgt tgtcggtctg attatcggtc tgggaccacg gtcccacttg tattgtcgat
     7741 cagactatca gcgtgagact acgattccat caatgcctgt caagggcaag tattgacatg
     7801 tcgtcgtaac ctgtagaacg gagtaacctc ggtgtgcggt tgtatgcctg ctgtggattg
     7861 ctgctgtgtc ctgcttatcc acaacatttt gcgcacggtt atgtggacaa aatacctggt
     7921 tacccaggcc gtgccggcac gttaaccggg ctgcatccga tgcaagtgtg tcgctgtcga
     7981 cgcaagcggc cgca
//
