LOCUS       sequence-480220-        7998 bp    DNA     circular SYN 24-JUL-2025
DEFINITION  pMOBC360_VL: Vector Left (VL) 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 7998)
  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 7998)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 24, 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: POC1553
FEATURES             Location/Qualifiers
     source          1..7998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      237..825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     726..745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     979..996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1113..1134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1149..1154,1155..1172,1173..1179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1187..1203
                     /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     1192..1214
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1211..1227
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1211..1227
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1223..1546
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1240..1296
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1297..1314)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1297..1313)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1306..1328)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1522..1541)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1828..1930
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1931..2590
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1997..2016)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      3405..3624
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3715..4470
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4473..4723
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5049..6224
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6224..7195
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7268..7741
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
ORIGIN
        1 cactctgtgg tgcagtacct ctcacgactt ggtctcactc ctgagaccgg cctagcaggt
       61 gtgagtttgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
      121 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
      181 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
      241 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
      301 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
      361 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
      421 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
      481 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
      541 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
      601 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
      661 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
      721 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
      781 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
      841 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
      901 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
      961 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggatgagcg cccaatacgc
     1021 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     1081 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     1141 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     1201 caatttcaca caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc
     1261 gactctagag gatccccggg taccgagctc gaattcactg gccgtcgttt tacaacgtcg
     1321 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     1381 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     1441 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     1501 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     1561 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     1621 cagacaagct gtgacaagta ccacctggaa cgccaaaggt ctcaagacca agtcgtgaga
     1681 ggtactgcac cacgaagtgt gcggccgcct tcagcggccg cacgaataaa tacctgtgac
     1741 ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg ggaagccctg
     1801 ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca actttcacca
     1861 taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa
     1921 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca
     1981 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt
     2041 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc
     2101 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggagttcc gtatggcaat
     2161 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga
     2221 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct
     2281 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg
     2341 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga
     2401 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta
     2461 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga
     2521 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg
     2581 cggggcgtaa gagctggagc ggcgtaaccg tcgcacagga aggacagaga aagcgcggat
     2641 ctgggaagtg acggacagaa cggtcaggac ctggattggg gaggcggttg ccgccgctgc
     2701 tgctgacggt gtgacgttct ctgttccggt cacaccacat acgttccgcc attcctatgc
     2761 gatgcacatg ctgtatgccg gtataccgct gaaagttctg caaagcctga tgggacataa
     2821 gtccatcagt tcaacggaag tctacacgaa ggtttttgcg ctggatgtgg ctgcccggct
     2881 ccgggtgcag tttgcgatgc cggagtctga tgcggttgcg atgctgaaac aattatcctg
     2941 agaataaatg ccttggcctt tatatggaaa tgtggaactg agtggatatg ctgtttttgt
     3001 ctgttaaaca gagaagctgg ctgttatcca ctgagaagcg aacgaaacag tcgggaaaat
     3061 ctcccattat cgtagagatc cgcattatta atctcaggag cctgtgtagc gtttatagga
     3121 agtagtgttc tgtcatgatg cctgcaagcg gtaacgaaaa cgatttgaat atgccttcag
     3181 gaacaataga aatcttcgtg cggtgttacg ttgaagtgga gcggattatg tcagcaatgg
     3241 acagaacaac ctaatgaaca cagaaccatg atgtggtctg tccttttaca gccagtagtg
     3301 ctcgccgcag tcgagcgaca gggcgaagcc ctcgagtgag cgaggaagca ccagggaaca
     3361 gcacttatat attctgctta cacacgatgc ctgaaaaaac ttcccttggg gttatccact
     3421 tatccacggg gatattttta taattatttt ttttatagtt tttagatctt cttttttaga
     3481 gcgccttgta ggcctttatc catgctggtt ctagagaagg tgttgtgaca aattgccctt
     3541 tcagtgtgac aaatcaccct caaatgacag tcctgtctgt gacaaattgc ccttaaccct
     3601 gtgacaaatt gccctcagaa gaagctgttt tttcacaaag ttatccctgc ttattgactc
     3661 ttttttattt agtgtgacaa tctaaaaact tgtcacactt cacatggatc tgtcatggcg
     3721 gaaacagcgg ttatcaatca caagaaacgt aaaaatagcc cgcgaatcgt ccagtcaaac
     3781 gacctcactg aggcggcata tagtctctcc cgggatcaaa aacgtatgct gtatctgttc
     3841 gttgaccaga tcagaaaatc tgatggcacc ctacaggaac atgacggtat ctgcgagatc
     3901 catgttgcta aatatgctga aatattcgga ttgacctctg cggaagccag taaggatata
     3961 cggcaggcat tgaagagttt cgcggggaag gaagtggttt tttatcgccc tgaagaggat
     4021 gccggcgatg aaaaaggcta tgaatctttt ccttggttta tcaaacgtgc gcacagtcca
     4081 tccagagggc tttacagtgt acatatcaac ccatatctca ttcccttctt tatcgggtta
     4141 cagaaccggt ttacgcagtt tcggcttagt gaaacaaaag aaatcaccaa tccgtatgcc
     4201 atgcgtttat acgaatccct gtgtcagtat cgtaagccgg atggctcagg catcgtgtct
     4261 ctgaaaatcg actggatcat agagcgttac cagctgcctc aaagttacca gcgtatgcct
     4321 gacttccgcc gccgcttcct gcaggtctgt gttaatgaga tcaacagcag aactccaatg
     4381 cgcctctcat acattgagaa aaagaaaggc cgccagacga ctcatatcgt attttccttc
     4441 cgcgatatca cttccatgac gacaggatag tctgagggtt atctgtcaca gatttgaggg
     4501 tggttcgtca catttgttct gacctactga gggtaatttg tcacagtttt gctgtttcct
     4561 tcagcctgca tggattttct catacttttt gaactgtaat ttttaaggaa gccaaatttg
     4621 agggcagttt gtcacagttg atttccttct ctttcccttc gtcatgtgac ctgatatcgg
     4681 gggttagttc gtcatcattg atgagggttg attatcacag tttattactc tgaattggct
     4741 atccgcgtgt gtacctctac ctggagtttt tcccacggtg gatatttctt cttgcgctga
     4801 gcgtaagagc tatctgacag aacagttctt ctttgcttcc tcgccagttc gctcgctatg
     4861 ctcggttaca cggctgcggc gagcgctagt gataataagt gactgaggta tgtggtgttc
     4921 ttatctcctt ttgtagtgtt gctcttattt taaacaactt tgcggttttt tgatgacttt
     4981 gcgattttgt tgttgctttg cagtaaattg caagatttaa taaaaaaacg caaagcaatg
     5041 attaaaggat gttcagaatg aaactcatgg aaacacttaa ccagtgcata aacgctggtc
     5101 atgaaatgac gaaggctatc gccattgcac agtttaatga tgacagcccg gaagcgagga
     5161 aaataacccg gcgctggaga ataggtgaag cagcggattt agttggggtt tcttctcagg
     5221 ctatcagaga tgccgagaaa gcagggcgac taccgcaccc ggatatggaa attcgaggac
     5281 gggttgagca acgtgttggt tatacaattg aacaaattaa tcatatgcgt gatgtgtttg
     5341 gtacgcgatt gcgacgtgct gaggacgtat ttccaccggt gatcggggtt gctgcccata
     5401 aaggtggcgt ttacaaaacc tcagtttctg ttcatcttgc tcaggatctg gctctgaagg
     5461 ggctacgtgt tttgctcgtg gaaggtaacg acccccaggg aacagcctca atgtatcacg
     5521 gatgggtacc agatcttcat attcatgcag aagatactct cctgcctttc tatcttgggg
     5581 aaaaggacga tgtcacttat gcaataaagc ccacttgctg gccggggctt gacattattc
     5641 cttcctgtct ggctctgcac cgtattgaaa ctgagttaat gggcaaattt gatgaaggta
     5701 aactgcccac cgatccacac ctgatgctcc gactggccat tgaaactgtt gctcatgact
     5761 atgatgtcat agttattgac agcgcgccta acctgggtat cggcacgatt aatgtcgtat
     5821 gtgctgctga tgtgctgatt gttcccacgc ctgctgagtt gtttgactac acctccgcac
     5881 tgcagttttt cgatatgctt cgtgatctgc tcaagaacgt tgatcttaaa gggttcgagc
     5941 ctgatgtacg tattttgctt accaaataca gcaatagtaa tggctctcag tccccgtgga
     6001 tggaggagca aattcgggat gcctggggaa gcatggttct aaaaaatgtt gtacgtgaaa
     6061 cggatgaagt tggtaaaggt cagatccgga tgagaactgt ttttgaacag gccattgatc
     6121 aacgcagttc aactggtgcc tggagaaatg ctctttctat ttgggaacct gtctgcaatg
     6181 aaattttcga tcgtctgatt aaaccacgct gggagattag ataatgaagc gtgcgcctgt
     6241 tattccaaaa catacgctca atactcaacc ggttgaagat acttcgttat cgacaccagc
     6301 tgccccgatg gtggattcgt taattgcgcg cgtaggagta atggctcgcg gtaatgccat
     6361 tactttgcct gtatgtggtc gggatgtgaa gtttactctt gaagtgctcc ggggtgatag
     6421 tgttgagaaa acctctcggg tatggtcagg taatgaacgt gaccaggagc tgcttactga
     6481 ggacgcactg gatgatctca tcccttcttt tctactgact ggtcaacaga caccggcgtt
     6541 cggtcgaaga gtatctggtg tcatagaaat tgccgatggg agtcgccgtc gtaaagctgc
     6601 tgcacttacc gaaagtgatt atcgtgttct ggttggcgag ctggatgatg agcagatggc
     6661 tgcattatcc agattgggta acgattatcg cccaacaagt gcttatgaac gtggtcagcg
     6721 ttatgcaagc cgattgcaga atgaatttgc tggaaatatt tctgcgctgg ctgatgcgga
     6781 aaatatttca cgtaagatta ttacccgctg tatcaacacc gccaaattgc ctaaatcagt
     6841 tgttgctctt ttttctcacc ccggtgaact atctgcccgg tcaggtgatg cacttcaaaa
     6901 agcctttaca gataaagagg aattacttaa gcagcaggca tctaaccttc atgagcagaa
     6961 aaaagctggg gtgatatttg aagctgaaga agttatcact cttttaactt ctgtgcttaa
     7021 aacgtcatct gcatcaagaa ctagtttaag ctcacgacat cagtttgctc ctggagcgac
     7081 agtattgtat aagggcgata aaatggtgct taacctggac aggtcacgtg ttccaactga
     7141 gtgtatagag aaaattgagg ccattcttaa ggaacttgaa aagccagcac cctgatgcga
     7201 ccacgtttta gtctacgttt atctgtcttt acttaatgtc ctttgttaca ggccagaaag
     7261 cataactggc ctgaatattc tctctgggcc cactgttcca cttgtatcgt cggtctgata
     7321 atcagactgg gaccacggtc ccactcgtat cgtcggtctg attattagtc tgggaccacg
     7381 gtcccactcg tatcgtcggt ctgattatta gtctgggacc acggtcccac tcgtatcgtc
     7441 ggtctgataa tcagactggg accacggtcc cactcgtatc gtcggtctga ttattagtct
     7501 gggaccatgg tcccactcgt atcgtcggtc tgattattag tctgggacca cggtcccact
     7561 cgtatcgtcg gtctgattat tagtctggaa ccacggtccc actcgtatcg tcggtctgat
     7621 tattagtctg ggaccacggt cccactcgta tcgtcggtct gattattagt ctgggaccac
     7681 gatcccactc gtgttgtcgg tctgattatc ggtctgggac cacggtccca cttgtattgt
     7741 cgatcagact atcagcgtga gactacgatt ccatcaatgc ctgtcaaggg caagtattga
     7801 catgtcgtcg taacctgtag aacggagtaa cctcggtgtg cggttgtatg cctgctgtgg
     7861 attgctgctg tgtcctgctt atccacaaca ttttgcgcac ggttatgtgg acaaaatacc
     7921 tggttaccca ggccgtgccg gcacgttaac cgggctgcat ccgatgcaag tgtgtcgctg
     7981 tcgacgcaag cggccgca
//
