LOCUS       sequence-476592-        8000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMOBC360_VR: Vector Right (VR) 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 8000)
  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 8000)
  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: POC1554
FEATURES             Location/Qualifiers
     source          1..8000
                     /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        1830..1932
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1933..2592
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1999..2018)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      3407..3626
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3717..4472
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4475..4725
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5051..6226
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6226..7197
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7270..7743
                     /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 cttcagcggc cgcacgaata aatacctgtg
     1741 acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac cgggaagccc
     1801 tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc caactttcac
     1861 cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt ttcaggagct
     1921 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
     1981 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
     2041 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
     2101 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggagtt ccgtatggca
     2161 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
     2221 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
     2281 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
     2341 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
     2401 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat
     2461 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
     2521 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
     2581 ggcggggcgt aagagctgga gcggcgtaac cgtcgcacag gaaggacaga gaaagcgcgg
     2641 atctgggaag tgacggacag aacggtcagg acctggattg gggaggcggt tgccgccgct
     2701 gctgctgacg gtgtgacgtt ctctgttccg gtcacaccac atacgttccg ccattcctat
     2761 gcgatgcaca tgctgtatgc cggtataccg ctgaaagttc tgcaaagcct gatgggacat
     2821 aagtccatca gttcaacgga agtctacacg aaggtttttg cgctggatgt ggctgcccgg
     2881 ctccgggtgc agtttgcgat gccggagtct gatgcggttg cgatgctgaa acaattatcc
     2941 tgagaataaa tgccttggcc tttatatgga aatgtggaac tgagtggata tgctgttttt
     3001 gtctgttaaa cagagaagct ggctgttatc cactgagaag cgaacgaaac agtcgggaaa
     3061 atctcccatt atcgtagaga tccgcattat taatctcagg agcctgtgta gcgtttatag
     3121 gaagtagtgt tctgtcatga tgcctgcaag cggtaacgaa aacgatttga atatgccttc
     3181 aggaacaata gaaatcttcg tgcggtgtta cgttgaagtg gagcggatta tgtcagcaat
     3241 ggacagaaca acctaatgaa cacagaacca tgatgtggtc tgtcctttta cagccagtag
     3301 tgctcgccgc agtcgagcga cagggcgaag ccctcgagtg agcgaggaag caccagggaa
     3361 cagcacttat atattctgct tacacacgat gcctgaaaaa acttcccttg gggttatcca
     3421 cttatccacg gggatatttt tataattatt ttttttatag tttttagatc ttctttttta
     3481 gagcgccttg taggccttta tccatgctgg ttctagagaa ggtgttgtga caaattgccc
     3541 tttcagtgtg acaaatcacc ctcaaatgac agtcctgtct gtgacaaatt gcccttaacc
     3601 ctgtgacaaa ttgccctcag aagaagctgt tttttcacaa agttatccct gcttattgac
     3661 tcttttttat ttagtgtgac aatctaaaaa cttgtcacac ttcacatgga tctgtcatgg
     3721 cggaaacagc ggttatcaat cacaagaaac gtaaaaatag cccgcgaatc gtccagtcaa
     3781 acgacctcac tgaggcggca tatagtctct cccgggatca aaaacgtatg ctgtatctgt
     3841 tcgttgacca gatcagaaaa tctgatggca ccctacagga acatgacggt atctgcgaga
     3901 tccatgttgc taaatatgct gaaatattcg gattgacctc tgcggaagcc agtaaggata
     3961 tacggcaggc attgaagagt ttcgcgggga aggaagtggt tttttatcgc cctgaagagg
     4021 atgccggcga tgaaaaaggc tatgaatctt ttccttggtt tatcaaacgt gcgcacagtc
     4081 catccagagg gctttacagt gtacatatca acccatatct cattcccttc tttatcgggt
     4141 tacagaaccg gtttacgcag tttcggctta gtgaaacaaa agaaatcacc aatccgtatg
     4201 ccatgcgttt atacgaatcc ctgtgtcagt atcgtaagcc ggatggctca ggcatcgtgt
     4261 ctctgaaaat cgactggatc atagagcgtt accagctgcc tcaaagttac cagcgtatgc
     4321 ctgacttccg ccgccgcttc ctgcaggtct gtgttaatga gatcaacagc agaactccaa
     4381 tgcgcctctc atacattgag aaaaagaaag gccgccagac gactcatatc gtattttcct
     4441 tccgcgatat cacttccatg acgacaggat agtctgaggg ttatctgtca cagatttgag
     4501 ggtggttcgt cacatttgtt ctgacctact gagggtaatt tgtcacagtt ttgctgtttc
     4561 cttcagcctg catggatttt ctcatacttt ttgaactgta atttttaagg aagccaaatt
     4621 tgagggcagt ttgtcacagt tgatttcctt ctctttccct tcgtcatgtg acctgatatc
     4681 gggggttagt tcgtcatcat tgatgagggt tgattatcac agtttattac tctgaattgg
     4741 ctatccgcgt gtgtacctct acctggagtt tttcccacgg tggatatttc ttcttgcgct
     4801 gagcgtaaga gctatctgac agaacagttc ttctttgctt cctcgccagt tcgctcgcta
     4861 tgctcggtta cacggctgcg gcgagcgcta gtgataataa gtgactgagg tatgtggtgt
     4921 tcttatctcc ttttgtagtg ttgctcttat tttaaacaac tttgcggttt tttgatgact
     4981 ttgcgatttt gttgttgctt tgcagtaaat tgcaagattt aataaaaaaa cgcaaagcaa
     5041 tgattaaagg atgttcagaa tgaaactcat ggaaacactt aaccagtgca taaacgctgg
     5101 tcatgaaatg acgaaggcta tcgccattgc acagtttaat gatgacagcc cggaagcgag
     5161 gaaaataacc cggcgctgga gaataggtga agcagcggat ttagttgggg tttcttctca
     5221 ggctatcaga gatgccgaga aagcagggcg actaccgcac ccggatatgg aaattcgagg
     5281 acgggttgag caacgtgttg gttatacaat tgaacaaatt aatcatatgc gtgatgtgtt
     5341 tggtacgcga ttgcgacgtg ctgaggacgt atttccaccg gtgatcgggg ttgctgccca
     5401 taaaggtggc gtttacaaaa cctcagtttc tgttcatctt gctcaggatc tggctctgaa
     5461 ggggctacgt gttttgctcg tggaaggtaa cgacccccag ggaacagcct caatgtatca
     5521 cggatgggta ccagatcttc atattcatgc agaagatact ctcctgcctt tctatcttgg
     5581 ggaaaaggac gatgtcactt atgcaataaa gcccacttgc tggccggggc ttgacattat
     5641 tccttcctgt ctggctctgc accgtattga aactgagtta atgggcaaat ttgatgaagg
     5701 taaactgccc accgatccac acctgatgct ccgactggcc attgaaactg ttgctcatga
     5761 ctatgatgtc atagttattg acagcgcgcc taacctgggt atcggcacga ttaatgtcgt
     5821 atgtgctgct gatgtgctga ttgttcccac gcctgctgag ttgtttgact acacctccgc
     5881 actgcagttt ttcgatatgc ttcgtgatct gctcaagaac gttgatctta aagggttcga
     5941 gcctgatgta cgtattttgc ttaccaaata cagcaatagt aatggctctc agtccccgtg
     6001 gatggaggag caaattcggg atgcctgggg aagcatggtt ctaaaaaatg ttgtacgtga
     6061 aacggatgaa gttggtaaag gtcagatccg gatgagaact gtttttgaac aggccattga
     6121 tcaacgcagt tcaactggtg cctggagaaa tgctctttct atttgggaac ctgtctgcaa
     6181 tgaaattttc gatcgtctga ttaaaccacg ctgggagatt agataatgaa gcgtgcgcct
     6241 gttattccaa aacatacgct caatactcaa ccggttgaag atacttcgtt atcgacacca
     6301 gctgccccga tggtggattc gttaattgcg cgcgtaggag taatggctcg cggtaatgcc
     6361 attactttgc ctgtatgtgg tcgggatgtg aagtttactc ttgaagtgct ccggggtgat
     6421 agtgttgaga aaacctctcg ggtatggtca ggtaatgaac gtgaccagga gctgcttact
     6481 gaggacgcac tggatgatct catcccttct tttctactga ctggtcaaca gacaccggcg
     6541 ttcggtcgaa gagtatctgg tgtcatagaa attgccgatg ggagtcgccg tcgtaaagct
     6601 gctgcactta ccgaaagtga ttatcgtgtt ctggttggcg agctggatga tgagcagatg
     6661 gctgcattat ccagattggg taacgattat cgcccaacaa gtgcttatga acgtggtcag
     6721 cgttatgcaa gccgattgca gaatgaattt gctggaaata tttctgcgct ggctgatgcg
     6781 gaaaatattt cacgtaagat tattacccgc tgtatcaaca ccgccaaatt gcctaaatca
     6841 gttgttgctc ttttttctca ccccggtgaa ctatctgccc ggtcaggtga tgcacttcaa
     6901 aaagccttta cagataaaga ggaattactt aagcagcagg catctaacct tcatgagcag
     6961 aaaaaagctg gggtgatatt tgaagctgaa gaagttatca ctcttttaac ttctgtgctt
     7021 aaaacgtcat ctgcatcaag aactagttta agctcacgac atcagtttgc tcctggagcg
     7081 acagtattgt ataagggcga taaaatggtg cttaacctgg acaggtcacg tgttccaact
     7141 gagtgtatag agaaaattga ggccattctt aaggaacttg aaaagccagc accctgatgc
     7201 gaccacgttt tagtctacgt ttatctgtct ttacttaatg tcctttgtta caggccagaa
     7261 agcataactg gcctgaatat tctctctggg cccactgttc cacttgtatc gtcggtctga
     7321 taatcagact gggaccacgg tcccactcgt atcgtcggtc tgattattag tctgggacca
     7381 cggtcccact cgtatcgtcg gtctgattat tagtctggga ccacggtccc actcgtatcg
     7441 tcggtctgat aatcagactg ggaccacggt cccactcgta tcgtcggtct gattattagt
     7501 ctgggaccat ggtcccactc gtatcgtcgg tctgattatt agtctgggac cacggtccca
     7561 ctcgtatcgt cggtctgatt attagtctgg aaccacggtc ccactcgtat cgtcggtctg
     7621 attattagtc tgggaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc
     7681 acgatcccac tcgtgttgtc ggtctgatta tcggtctggg accacggtcc cacttgtatt
     7741 gtcgatcaga ctatcagcgt gagactacga ttccatcaat gcctgtcaag ggcaagtatt
     7801 gacatgtcgt cgtaacctgt agaacggagt aacctcggtg tgcggttgta tgcctgctgt
     7861 ggattgctgc tgtgtcctgc ttatccacaa cattttgcgc acggttatgt ggacaaaata
     7921 cctggttacc caggccgtgc cggcacgtta accgggctgc atccgatgca agtgtgtcgc
     7981 tgtcgacgca agcggccgca
//
