LOCUS       sequence-465755-        8023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMOBK360_VL: Vector Left (VL) 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 8023)
  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 8023)
  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: POC1518
FEATURES             Location/Qualifiers
     source          1..8023
                     /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        1716..1820
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1821..2615
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3430..3649
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3740..4495
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4498..4748
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5074..6249
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6249..7220
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7293..7766
                     /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 tcgtgcgcgg aacccctatt tgtttatttt
     1741 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1801 aatattgaaa aaggaagagt atggctaaaa tgagaatatc accggaattg aaaaaactga
     1861 tcgaaaaata ccgctgcgta aaagatacgg aaggaatgtc tcctgctaag gtatataagc
     1921 tggtgggaga aaatgaaaac ctatatttaa aaatgacgga cagccggtat aaagggacca
     1981 cctatgatgt ggaacgggaa aaggacatga tgctatggct ggaaggaaag ctgcctgttc
     2041 caaaggtcct gcactttgaa cggcatgatg gctggagcaa tctgctcatg agtgaggccg
     2101 atggcgtcct ttgctcggaa gagtatgaag atgaacaaag ccctgaaaag attatcgagc
     2161 tgtatgcgga gtgcatcagg ctctttcact ccatcgacat atcggattgt ccctatacga
     2221 atagcttaga cagccgctta gccgaattgg attacttact gaataacgat ctggccgatg
     2281 tggattgcga aaactgggaa gaggacactc catttaaaga tccgcgcgag ctgtatgatt
     2341 ttttaaagac ggaaaagccc gaagaggaac ttgtcttttc ccacggcgac ctgggagaca
     2401 gcaacatctt tgtgaaagat ggcaaagtaa gtggctttat tgatcttggg agaagcggca
     2461 gggcggacaa gtggtatgac attgccttct gcgtccggtc gctcagggag gatatcgggg
     2521 aagaacagta tgtcgagcta ttttttgact tactggggat caagcctgat tgggagaaaa
     2581 taaaatatta tattttactg gatgaattgt tttaggagct ggagcggcgt aaccgtcgca
     2641 caggaaggac agagaaagcg cggatctggg aagtgacgga cagaacggtc aggacctgga
     2701 ttggggaggc ggttgccgcc gctgctgctg acggtgtgac gttctctgtt ccggtcacac
     2761 cacatacgtt ccgccattcc tatgcgatgc acatgctgta tgccggtata ccgctgaaag
     2821 ttctgcaaag cctgatggga cataagtcca tcagttcaac ggaagtctac acgaaggttt
     2881 ttgcgctgga tgtggctgcc cggctccggg tgcagtttgc gatgccggag tctgatgcgg
     2941 ttgcgatgct gaaacaatta tcctgagaat aaatgccttg gcctttatat ggaaatgtgg
     3001 aactgagtgg atatgctgtt tttgtctgtt aaacagagaa gctggctgtt atccactgag
     3061 aagcgaacga aacagtcggg aaaatctccc attatcgtag agatccgcat tattaatctc
     3121 aggagcctgt gtagcgttta taggaagtag tgttctgtca tgatgcctgc aagcggtaac
     3181 gaaaacgatt tgaatatgcc ttcaggaaca atagaaatct tcgtgcggtg ttacgttgaa
     3241 gtggagcgga ttatgtcagc aatggacaga acaacctaat gaacacagaa ccatgatgtg
     3301 gtctgtcctt ttacagccag tagtgctcgc cgcagtcgag cgacagggcg aagccctcga
     3361 gtgagcgagg aagcaccagg gaacagcact tatatattct gcttacacac gatgcctgaa
     3421 aaaacttccc ttggggttat ccacttatcc acggggatat ttttataatt atttttttta
     3481 tagtttttag atcttctttt ttagagcgcc ttgtaggcct ttatccatgc tggttctaga
     3541 gaaggtgttg tgacaaattg ccctttcagt gtgacaaatc accctcaaat gacagtcctg
     3601 tctgtgacaa attgccctta accctgtgac aaattgccct cagaagaagc tgttttttca
     3661 caaagttatc cctgcttatt gactcttttt tatttagtgt gacaatctaa aaacttgtca
     3721 cacttcacat ggatctgtca tggcggaaac agcggttatc aatcacaaga aacgtaaaaa
     3781 tagcccgcga atcgtccagt caaacgacct cactgaggcg gcatatagtc tctcccggga
     3841 tcaaaaacgt atgctgtatc tgttcgttga ccagatcaga aaatctgatg gcaccctaca
     3901 ggaacatgac ggtatctgcg agatccatgt tgctaaatat gctgaaatat tcggattgac
     3961 ctctgcggaa gccagtaagg atatacggca ggcattgaag agtttcgcgg ggaaggaagt
     4021 ggttttttat cgccctgaag aggatgccgg cgatgaaaaa ggctatgaat cttttccttg
     4081 gtttatcaaa cgtgcgcaca gtccatccag agggctttac agtgtacata tcaacccata
     4141 tctcattccc ttctttatcg ggttacagaa ccggtttacg cagtttcggc ttagtgaaac
     4201 aaaagaaatc accaatccgt atgccatgcg tttatacgaa tccctgtgtc agtatcgtaa
     4261 gccggatggc tcaggcatcg tgtctctgaa aatcgactgg atcatagagc gttaccagct
     4321 gcctcaaagt taccagcgta tgcctgactt ccgccgccgc ttcctgcagg tctgtgttaa
     4381 tgagatcaac agcagaactc caatgcgcct ctcatacatt gagaaaaaga aaggccgcca
     4441 gacgactcat atcgtatttt ccttccgcga tatcacttcc atgacgacag gatagtctga
     4501 gggttatctg tcacagattt gagggtggtt cgtcacattt gttctgacct actgagggta
     4561 atttgtcaca gttttgctgt ttccttcagc ctgcatggat tttctcatac tttttgaact
     4621 gtaattttta aggaagccaa atttgagggc agtttgtcac agttgatttc cttctctttc
     4681 ccttcgtcat gtgacctgat atcgggggtt agttcgtcat cattgatgag ggttgattat
     4741 cacagtttat tactctgaat tggctatccg cgtgtgtacc tctacctgga gtttttccca
     4801 cggtggatat ttcttcttgc gctgagcgta agagctatct gacagaacag ttcttctttg
     4861 cttcctcgcc agttcgctcg ctatgctcgg ttacacggct gcggcgagcg ctagtgataa
     4921 taagtgactg aggtatgtgg tgttcttatc tccttttgta gtgttgctct tattttaaac
     4981 aactttgcgg ttttttgatg actttgcgat tttgttgttg ctttgcagta aattgcaaga
     5041 tttaataaaa aaacgcaaag caatgattaa aggatgttca gaatgaaact catggaaaca
     5101 cttaaccagt gcataaacgc tggtcatgaa atgacgaagg ctatcgccat tgcacagttt
     5161 aatgatgaca gcccggaagc gaggaaaata acccggcgct ggagaatagg tgaagcagcg
     5221 gatttagttg gggtttcttc tcaggctatc agagatgccg agaaagcagg gcgactaccg
     5281 cacccggata tggaaattcg aggacgggtt gagcaacgtg ttggttatac aattgaacaa
     5341 attaatcata tgcgtgatgt gtttggtacg cgattgcgac gtgctgagga cgtatttcca
     5401 ccggtgatcg gggttgctgc ccataaaggt ggcgtttaca aaacctcagt ttctgttcat
     5461 cttgctcagg atctggctct gaaggggcta cgtgttttgc tcgtggaagg taacgacccc
     5521 cagggaacag cctcaatgta tcacggatgg gtaccagatc ttcatattca tgcagaagat
     5581 actctcctgc ctttctatct tggggaaaag gacgatgtca cttatgcaat aaagcccact
     5641 tgctggccgg ggcttgacat tattccttcc tgtctggctc tgcaccgtat tgaaactgag
     5701 ttaatgggca aatttgatga aggtaaactg cccaccgatc cacacctgat gctccgactg
     5761 gccattgaaa ctgttgctca tgactatgat gtcatagtta ttgacagcgc gcctaacctg
     5821 ggtatcggca cgattaatgt cgtatgtgct gctgatgtgc tgattgttcc cacgcctgct
     5881 gagttgtttg actacacctc cgcactgcag tttttcgata tgcttcgtga tctgctcaag
     5941 aacgttgatc ttaaagggtt cgagcctgat gtacgtattt tgcttaccaa atacagcaat
     6001 agtaatggct ctcagtcccc gtggatggag gagcaaattc gggatgcctg gggaagcatg
     6061 gttctaaaaa atgttgtacg tgaaacggat gaagttggta aaggtcagat ccggatgaga
     6121 actgtttttg aacaggccat tgatcaacgc agttcaactg gtgcctggag aaatgctctt
     6181 tctatttggg aacctgtctg caatgaaatt ttcgatcgtc tgattaaacc acgctgggag
     6241 attagataat gaagcgtgcg cctgttattc caaaacatac gctcaatact caaccggttg
     6301 aagatacttc gttatcgaca ccagctgccc cgatggtgga ttcgttaatt gcgcgcgtag
     6361 gagtaatggc tcgcggtaat gccattactt tgcctgtatg tggtcgggat gtgaagttta
     6421 ctcttgaagt gctccggggt gatagtgttg agaaaacctc tcgggtatgg tcaggtaatg
     6481 aacgtgacca ggagctgctt actgaggacg cactggatga tctcatccct tcttttctac
     6541 tgactggtca acagacaccg gcgttcggtc gaagagtatc tggtgtcata gaaattgccg
     6601 atgggagtcg ccgtcgtaaa gctgctgcac ttaccgaaag tgattatcgt gttctggttg
     6661 gcgagctgga tgatgagcag atggctgcat tatccagatt gggtaacgat tatcgcccaa
     6721 caagtgctta tgaacgtggt cagcgttatg caagccgatt gcagaatgaa tttgctggaa
     6781 atatttctgc gctggctgat gcggaaaata tttcacgtaa gattattacc cgctgtatca
     6841 acaccgccaa attgcctaaa tcagttgttg ctcttttttc tcaccccggt gaactatctg
     6901 cccggtcagg tgatgcactt caaaaagcct ttacagataa agaggaatta cttaagcagc
     6961 aggcatctaa ccttcatgag cagaaaaaag ctggggtgat atttgaagct gaagaagtta
     7021 tcactctttt aacttctgtg cttaaaacgt catctgcatc aagaactagt ttaagctcac
     7081 gacatcagtt tgctcctgga gcgacagtat tgtataaggg cgataaaatg gtgcttaacc
     7141 tggacaggtc acgtgttcca actgagtgta tagagaaaat tgaggccatt cttaaggaac
     7201 ttgaaaagcc agcaccctga tgcgaccacg ttttagtcta cgtttatctg tctttactta
     7261 atgtcctttg ttacaggcca gaaagcataa ctggcctgaa tattctctct gggcccactg
     7321 ttccacttgt atcgtcggtc tgataatcag actgggacca cggtcccact cgtatcgtcg
     7381 gtctgattat tagtctggga ccacggtccc actcgtatcg tcggtctgat tattagtctg
     7441 ggaccacggt cccactcgta tcgtcggtct gataatcaga ctgggaccac ggtcccactc
     7501 gtatcgtcgg tctgattatt agtctgggac catggtccca ctcgtatcgt cggtctgatt
     7561 attagtctgg gaccacggtc ccactcgtat cgtcggtctg attattagtc tggaaccacg
     7621 gtcccactcg tatcgtcggt ctgattatta gtctgggacc acggtcccac tcgtatcgtc
     7681 ggtctgatta ttagtctggg accacgatcc cactcgtgtt gtcggtctga ttatcggtct
     7741 gggaccacgg tcccacttgt attgtcgatc agactatcag cgtgagacta cgattccatc
     7801 aatgcctgtc aagggcaagt attgacatgt cgtcgtaacc tgtagaacgg agtaacctcg
     7861 gtgtgcggtt gtatgcctgc tgtggattgc tgctgtgtcc tgcttatcca caacattttg
     7921 cgcacggtta tgtggacaaa atacctggtt acccaggccg tgccggcacg ttaaccgggc
     7981 tgcatccgat gcaagtgtgt cgctgtcgac gcaagcggcc gca
//
