LOCUS       sequence-465765-        8029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMOBK360_StandVKL: Standard Vector (Kanamycin R) to be methylated by
            the M.Osp807II BsaI-associated switch methylase. Used for the 
            assembly using the methylation protection .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8029)
  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 8029)
  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: POC1551
FEATURES             Location/Qualifiers
     source          1..8029
                     /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        1722..1826
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1827..2621
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3436..3655
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3746..4501
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4504..4754
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5080..6255
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6255..7226
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7299..7772
                     /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 ctcaagactg agaccaagtc
     1681 gtgagaggta ctgcaccacg aagtgtgcgg ccgccttcgt gcgcggaacc cctatttgtt
     1741 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1801 ttcaataata ttgaaaaagg aagagtatgg ctaaaatgag aatatcaccg gaattgaaaa
     1861 aactgatcga aaaataccgc tgcgtaaaag atacggaagg aatgtctcct gctaaggtat
     1921 ataagctggt gggagaaaat gaaaacctat atttaaaaat gacggacagc cggtataaag
     1981 ggaccaccta tgatgtggaa cgggaaaagg acatgatgct atggctggaa ggaaagctgc
     2041 ctgttccaaa ggtcctgcac tttgaacggc atgatggctg gagcaatctg ctcatgagtg
     2101 aggccgatgg cgtcctttgc tcggaagagt atgaagatga acaaagccct gaaaagatta
     2161 tcgagctgta tgcggagtgc atcaggctct ttcactccat cgacatatcg gattgtccct
     2221 atacgaatag cttagacagc cgcttagccg aattggatta cttactgaat aacgatctgg
     2281 ccgatgtgga ttgcgaaaac tgggaagagg acactccatt taaagatccg cgcgagctgt
     2341 atgatttttt aaagacggaa aagcccgaag aggaacttgt cttttcccac ggcgacctgg
     2401 gagacagcaa catctttgtg aaagatggca aagtaagtgg ctttattgat cttgggagaa
     2461 gcggcagggc ggacaagtgg tatgacattg ccttctgcgt ccggtcgctc agggaggata
     2521 tcggggaaga acagtatgtc gagctatttt ttgacttact ggggatcaag cctgattggg
     2581 agaaaataaa atattatatt ttactggatg aattgtttta ggagctggag cggcgtaacc
     2641 gtcgcacagg aaggacagag aaagcgcgga tctgggaagt gacggacaga acggtcagga
     2701 cctggattgg ggaggcggtt gccgccgctg ctgctgacgg tgtgacgttc tctgttccgg
     2761 tcacaccaca tacgttccgc cattcctatg cgatgcacat gctgtatgcc ggtataccgc
     2821 tgaaagttct gcaaagcctg atgggacata agtccatcag ttcaacggaa gtctacacga
     2881 aggtttttgc gctggatgtg gctgcccggc tccgggtgca gtttgcgatg ccggagtctg
     2941 atgcggttgc gatgctgaaa caattatcct gagaataaat gccttggcct ttatatggaa
     3001 atgtggaact gagtggatat gctgtttttg tctgttaaac agagaagctg gctgttatcc
     3061 actgagaagc gaacgaaaca gtcgggaaaa tctcccatta tcgtagagat ccgcattatt
     3121 aatctcagga gcctgtgtag cgtttatagg aagtagtgtt ctgtcatgat gcctgcaagc
     3181 ggtaacgaaa acgatttgaa tatgccttca ggaacaatag aaatcttcgt gcggtgttac
     3241 gttgaagtgg agcggattat gtcagcaatg gacagaacaa cctaatgaac acagaaccat
     3301 gatgtggtct gtccttttac agccagtagt gctcgccgca gtcgagcgac agggcgaagc
     3361 cctcgagtga gcgaggaagc accagggaac agcacttata tattctgctt acacacgatg
     3421 cctgaaaaaa cttcccttgg ggttatccac ttatccacgg ggatattttt ataattattt
     3481 tttttatagt ttttagatct tcttttttag agcgccttgt aggcctttat ccatgctggt
     3541 tctagagaag gtgttgtgac aaattgccct ttcagtgtga caaatcaccc tcaaatgaca
     3601 gtcctgtctg tgacaaattg cccttaaccc tgtgacaaat tgccctcaga agaagctgtt
     3661 ttttcacaaa gttatccctg cttattgact cttttttatt tagtgtgaca atctaaaaac
     3721 ttgtcacact tcacatggat ctgtcatggc ggaaacagcg gttatcaatc acaagaaacg
     3781 taaaaatagc ccgcgaatcg tccagtcaaa cgacctcact gaggcggcat atagtctctc
     3841 ccgggatcaa aaacgtatgc tgtatctgtt cgttgaccag atcagaaaat ctgatggcac
     3901 cctacaggaa catgacggta tctgcgagat ccatgttgct aaatatgctg aaatattcgg
     3961 attgacctct gcggaagcca gtaaggatat acggcaggca ttgaagagtt tcgcggggaa
     4021 ggaagtggtt ttttatcgcc ctgaagagga tgccggcgat gaaaaaggct atgaatcttt
     4081 tccttggttt atcaaacgtg cgcacagtcc atccagaggg ctttacagtg tacatatcaa
     4141 cccatatctc attcccttct ttatcgggtt acagaaccgg tttacgcagt ttcggcttag
     4201 tgaaacaaaa gaaatcacca atccgtatgc catgcgttta tacgaatccc tgtgtcagta
     4261 tcgtaagccg gatggctcag gcatcgtgtc tctgaaaatc gactggatca tagagcgtta
     4321 ccagctgcct caaagttacc agcgtatgcc tgacttccgc cgccgcttcc tgcaggtctg
     4381 tgttaatgag atcaacagca gaactccaat gcgcctctca tacattgaga aaaagaaagg
     4441 ccgccagacg actcatatcg tattttcctt ccgcgatatc acttccatga cgacaggata
     4501 gtctgagggt tatctgtcac agatttgagg gtggttcgtc acatttgttc tgacctactg
     4561 agggtaattt gtcacagttt tgctgtttcc ttcagcctgc atggattttc tcatactttt
     4621 tgaactgtaa tttttaagga agccaaattt gagggcagtt tgtcacagtt gatttccttc
     4681 tctttccctt cgtcatgtga cctgatatcg ggggttagtt cgtcatcatt gatgagggtt
     4741 gattatcaca gtttattact ctgaattggc tatccgcgtg tgtacctcta cctggagttt
     4801 ttcccacggt ggatatttct tcttgcgctg agcgtaagag ctatctgaca gaacagttct
     4861 tctttgcttc ctcgccagtt cgctcgctat gctcggttac acggctgcgg cgagcgctag
     4921 tgataataag tgactgaggt atgtggtgtt cttatctcct tttgtagtgt tgctcttatt
     4981 ttaaacaact ttgcggtttt ttgatgactt tgcgattttg ttgttgcttt gcagtaaatt
     5041 gcaagattta ataaaaaaac gcaaagcaat gattaaagga tgttcagaat gaaactcatg
     5101 gaaacactta accagtgcat aaacgctggt catgaaatga cgaaggctat cgccattgca
     5161 cagtttaatg atgacagccc ggaagcgagg aaaataaccc ggcgctggag aataggtgaa
     5221 gcagcggatt tagttggggt ttcttctcag gctatcagag atgccgagaa agcagggcga
     5281 ctaccgcacc cggatatgga aattcgagga cgggttgagc aacgtgttgg ttatacaatt
     5341 gaacaaatta atcatatgcg tgatgtgttt ggtacgcgat tgcgacgtgc tgaggacgta
     5401 tttccaccgg tgatcggggt tgctgcccat aaaggtggcg tttacaaaac ctcagtttct
     5461 gttcatcttg ctcaggatct ggctctgaag gggctacgtg ttttgctcgt ggaaggtaac
     5521 gacccccagg gaacagcctc aatgtatcac ggatgggtac cagatcttca tattcatgca
     5581 gaagatactc tcctgccttt ctatcttggg gaaaaggacg atgtcactta tgcaataaag
     5641 cccacttgct ggccggggct tgacattatt ccttcctgtc tggctctgca ccgtattgaa
     5701 actgagttaa tgggcaaatt tgatgaaggt aaactgccca ccgatccaca cctgatgctc
     5761 cgactggcca ttgaaactgt tgctcatgac tatgatgtca tagttattga cagcgcgcct
     5821 aacctgggta tcggcacgat taatgtcgta tgtgctgctg atgtgctgat tgttcccacg
     5881 cctgctgagt tgtttgacta cacctccgca ctgcagtttt tcgatatgct tcgtgatctg
     5941 ctcaagaacg ttgatcttaa agggttcgag cctgatgtac gtattttgct taccaaatac
     6001 agcaatagta atggctctca gtccccgtgg atggaggagc aaattcggga tgcctgggga
     6061 agcatggttc taaaaaatgt tgtacgtgaa acggatgaag ttggtaaagg tcagatccgg
     6121 atgagaactg tttttgaaca ggccattgat caacgcagtt caactggtgc ctggagaaat
     6181 gctctttcta tttgggaacc tgtctgcaat gaaattttcg atcgtctgat taaaccacgc
     6241 tgggagatta gataatgaag cgtgcgcctg ttattccaaa acatacgctc aatactcaac
     6301 cggttgaaga tacttcgtta tcgacaccag ctgccccgat ggtggattcg ttaattgcgc
     6361 gcgtaggagt aatggctcgc ggtaatgcca ttactttgcc tgtatgtggt cgggatgtga
     6421 agtttactct tgaagtgctc cggggtgata gtgttgagaa aacctctcgg gtatggtcag
     6481 gtaatgaacg tgaccaggag ctgcttactg aggacgcact ggatgatctc atcccttctt
     6541 ttctactgac tggtcaacag acaccggcgt tcggtcgaag agtatctggt gtcatagaaa
     6601 ttgccgatgg gagtcgccgt cgtaaagctg ctgcacttac cgaaagtgat tatcgtgttc
     6661 tggttggcga gctggatgat gagcagatgg ctgcattatc cagattgggt aacgattatc
     6721 gcccaacaag tgcttatgaa cgtggtcagc gttatgcaag ccgattgcag aatgaatttg
     6781 ctggaaatat ttctgcgctg gctgatgcgg aaaatatttc acgtaagatt attacccgct
     6841 gtatcaacac cgccaaattg cctaaatcag ttgttgctct tttttctcac cccggtgaac
     6901 tatctgcccg gtcaggtgat gcacttcaaa aagcctttac agataaagag gaattactta
     6961 agcagcaggc atctaacctt catgagcaga aaaaagctgg ggtgatattt gaagctgaag
     7021 aagttatcac tcttttaact tctgtgctta aaacgtcatc tgcatcaaga actagtttaa
     7081 gctcacgaca tcagtttgct cctggagcga cagtattgta taagggcgat aaaatggtgc
     7141 ttaacctgga caggtcacgt gttccaactg agtgtataga gaaaattgag gccattctta
     7201 aggaacttga aaagccagca ccctgatgcg accacgtttt agtctacgtt tatctgtctt
     7261 tacttaatgt cctttgttac aggccagaaa gcataactgg cctgaatatt ctctctgggc
     7321 ccactgttcc acttgtatcg tcggtctgat aatcagactg ggaccacggt cccactcgta
     7381 tcgtcggtct gattattagt ctgggaccac ggtcccactc gtatcgtcgg tctgattatt
     7441 agtctgggac cacggtccca ctcgtatcgt cggtctgata atcagactgg gaccacggtc
     7501 ccactcgtat cgtcggtctg attattagtc tgggaccatg gtcccactcg tatcgtcggt
     7561 ctgattatta gtctgggacc acggtcccac tcgtatcgtc ggtctgatta ttagtctgga
     7621 accacggtcc cactcgtatc gtcggtctga ttattagtct gggaccacgg tcccactcgt
     7681 atcgtcggtc tgattattag tctgggacca cgatcccact cgtgttgtcg gtctgattat
     7741 cggtctggga ccacggtccc acttgtattg tcgatcagac tatcagcgtg agactacgat
     7801 tccatcaatg cctgtcaagg gcaagtattg acatgtcgtc gtaacctgta gaacggagta
     7861 acctcggtgt gcggttgtat gcctgctgtg gattgctgct gtgtcctgct tatccacaac
     7921 attttgcgca cggttatgtg gacaaaatac ctggttaccc aggccgtgcc ggcacgttaa
     7981 ccgggctgca tccgatgcaa gtgtgtcgct gtcgacgcaa gcggccgca
//
