LOCUS       Exported                8003 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  POC12398, MetClo assembly vector with F replication origin and 
            kanamycin resistance for BsaI-based MetClo, adaptor sequence type 
            a-b.
ACCESSION   .
VERSION     .
KEYWORDS    pMXBK_aFb
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8003)
  AUTHORS   Lin D, O'Callaghan CA
  TITLE     MetClo: methylase-assisted hierarchical DNA assembly using a single 
            type IIS restriction enzyme.
  JOURNAL   Nucleic Acids Res. 2018 Jul 9. pii: 5050631. doi: 
            10.1093/nar/gky596.
  PUBMED    29986052
REFERENCE   2  (bases 1 to 8003)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8003
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      224..812
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     713..732
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     966..983
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1100..1121
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1136..1166
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1174..1190
                     /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     1179..1201
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1198..1214
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1198..1214
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1210..1533
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1227..1283
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1284..1301)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1284..1300)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1293..1315)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1509..1528)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1696..1800
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1801..2595
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3410..3629
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3720..4475
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4478..4728
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5054..6229
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6229..7200
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7273..7746
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
ORIGIN
        1 cactctgtga cttggtctca aggtggagtg agaccggcct agcaggtgtg agtttgtcag
       61 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
      121 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
      181 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
      241 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
      301 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
      361 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
      421 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
      481 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
      541 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
      601 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
      661 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
      721 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
      781 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
      841 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
      901 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
      961 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     1021 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     1081 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     1141 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     1201 gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggtcgac tctagaggat
     1261 ccccgggtac cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     1321 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     1381 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     1441 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc
     1501 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     1561 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     1621 acaagtacca cctggaacgc caaaggtctc acgcttgcct gagaccaagt cacgaagtgt
     1681 gcggccgcct tcgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1741 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1801 atggctaaaa tgagaatatc accggaattg aaaaaactga tcgaaaaata ccgctgcgta
     1861 aaagatacgg aaggaatgtc tcctgctaag gtatataagc tggtgggaga aaatgaaaac
     1921 ctatatttaa aaatgacgga cagccggtat aaagggacca cctatgatgt ggaacgggaa
     1981 aaggacatga tgctatggct ggaaggaaag ctgcctgttc caaaggtcct gcactttgaa
     2041 cggcatgatg gctggagcaa tctgctcatg agtgaggccg atggcgtcct ttgctcggaa
     2101 gagtatgaag atgaacaaag ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg
     2161 ctctttcact ccatcgacat atcggattgt ccctatacga atagcttaga cagccgctta
     2221 gccgaattgg attacttact gaataacgat ctggccgatg tggattgcga aaactgggaa
     2281 gaggacactc catttaaaga tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc
     2341 gaagaggaac ttgtcttttc ccacggcgac ctgggagaca gcaacatctt tgtgaaagat
     2401 ggcaaagtaa gtggctttat tgatcttggg agaagcggca gggcggacaa gtggtatgac
     2461 attgccttct gcgtccggtc gctcagggag gatatcgggg aagaacagta tgtcgagcta
     2521 ttttttgact tactggggat caagcctgat tgggagaaaa taaaatatta tattttactg
     2581 gatgaattgt tttaggagct ggagcggcgt aaccgtcgca caggaaggac agagaaagcg
     2641 cggatctggg aagtgacgga cagaacggtc aggacctgga ttggggaggc ggttgccgcc
     2701 gctgctgctg acggtgtgac gttctctgtt ccggtcacac cacatacgtt ccgccattcc
     2761 tatgcgatgc acatgctgta tgccggtata ccgctgaaag ttctgcaaag cctgatggga
     2821 cataagtcca tcagttcaac ggaagtctac acgaaggttt ttgcgctgga tgtggctgcc
     2881 cggctccggg tgcagtttgc gatgccggag tctgatgcgg ttgcgatgct gaaacaatta
     2941 tcctgagaat aaatgccttg gcctttatat ggaaatgtgg aactgagtgg atatgctgtt
     3001 tttgtctgtt aaacagagaa gctggctgtt atccactgag aagcgaacga aacagtcggg
     3061 aaaatctccc attatcgtag agatccgcat tattaatctc aggagcctgt gtagcgttta
     3121 taggaagtag tgttctgtca tgatgcctgc aagcggtaac gaaaacgatt tgaatatgcc
     3181 ttcaggaaca atagaaatct tcgtgcggtg ttacgttgaa gtggagcgga ttatgtcagc
     3241 aatggacaga acaacctaat gaacacagaa ccatgatgtg gtctgtcctt ttacagccag
     3301 tagtgctcgc cgcagtcgag cgacagggcg aagccctcga gtgagcgagg aagcaccagg
     3361 gaacagcact tatatattct gcttacacac gatgcctgaa aaaacttccc ttggggttat
     3421 ccacttatcc acggggatat ttttataatt atttttttta tagtttttag atcttctttt
     3481 ttagagcgcc ttgtaggcct ttatccatgc tggttctaga gaaggtgttg tgacaaattg
     3541 ccctttcagt gtgacaaatc accctcaaat gacagtcctg tctgtgacaa attgccctta
     3601 accctgtgac aaattgccct cagaagaagc tgttttttca caaagttatc cctgcttatt
     3661 gactcttttt tatttagtgt gacaatctaa aaacttgtca cacttcacat ggatctgtca
     3721 tggcggaaac agcggttatc aatcacaaga aacgtaaaaa tagcccgcga atcgtccagt
     3781 caaacgacct cactgaggcg gcatatagtc tctcccggga tcaaaaacgt atgctgtatc
     3841 tgttcgttga ccagatcaga aaatctgatg gcaccctaca ggaacatgac ggtatctgcg
     3901 agatccatgt tgctaaatat gctgaaatat tcggattgac ctctgcggaa gccagtaagg
     3961 atatacggca ggcattgaag agtttcgcgg ggaaggaagt ggttttttat cgccctgaag
     4021 aggatgccgg cgatgaaaaa ggctatgaat cttttccttg gtttatcaaa cgtgcgcaca
     4081 gtccatccag agggctttac agtgtacata tcaacccata tctcattccc ttctttatcg
     4141 ggttacagaa ccggtttacg cagtttcggc ttagtgaaac aaaagaaatc accaatccgt
     4201 atgccatgcg tttatacgaa tccctgtgtc agtatcgtaa gccggatggc tcaggcatcg
     4261 tgtctctgaa aatcgactgg atcatagagc gttaccagct gcctcaaagt taccagcgta
     4321 tgcctgactt ccgccgccgc ttcctgcagg tctgtgttaa tgagatcaac agcagaactc
     4381 caatgcgcct ctcatacatt gagaaaaaga aaggccgcca gacgactcat atcgtatttt
     4441 ccttccgcga tatcacttcc atgacgacag gatagtctga gggttatctg tcacagattt
     4501 gagggtggtt cgtcacattt gttctgacct actgagggta atttgtcaca gttttgctgt
     4561 ttccttcagc ctgcatggat tttctcatac tttttgaact gtaattttta aggaagccaa
     4621 atttgagggc agtttgtcac agttgatttc cttctctttc ccttcgtcat gtgacctgat
     4681 atcgggggtt agttcgtcat cattgatgag ggttgattat cacagtttat tactctgaat
     4741 tggctatccg cgtgtgtacc tctacctgga gtttttccca cggtggatat ttcttcttgc
     4801 gctgagcgta agagctatct gacagaacag ttcttctttg cttcctcgcc agttcgctcg
     4861 ctatgctcgg ttacacggct gcggcgagcg ctagtgataa taagtgactg aggtatgtgg
     4921 tgttcttatc tccttttgta gtgttgctct tattttaaac aactttgcgg ttttttgatg
     4981 actttgcgat tttgttgttg ctttgcagta aattgcaaga tttaataaaa aaacgcaaag
     5041 caatgattaa aggatgttca gaatgaaact catggaaaca cttaaccagt gcataaacgc
     5101 tggtcatgaa atgacgaagg ctatcgccat tgcacagttt aatgatgaca gcccggaagc
     5161 gaggaaaata acccggcgct ggagaatagg tgaagcagcg gatttagttg gggtttcttc
     5221 tcaggctatc agagatgccg agaaagcagg gcgactaccg cacccggata tggaaattcg
     5281 aggacgggtt gagcaacgtg ttggttatac aattgaacaa attaatcata tgcgtgatgt
     5341 gtttggtacg cgattgcgac gtgctgagga cgtatttcca ccggtgatcg gggttgctgc
     5401 ccataaaggt ggcgtttaca aaacctcagt ttctgttcat cttgctcagg atctggctct
     5461 gaaggggcta cgtgttttgc tcgtggaagg taacgacccc cagggaacag cctcaatgta
     5521 tcacggatgg gtaccagatc ttcatattca tgcagaagat actctcctgc ctttctatct
     5581 tggggaaaag gacgatgtca cttatgcaat aaagcccact tgctggccgg ggcttgacat
     5641 tattccttcc tgtctggctc tgcaccgtat tgaaactgag ttaatgggca aatttgatga
     5701 aggtaaactg cccaccgatc cacacctgat gctccgactg gccattgaaa ctgttgctca
     5761 tgactatgat gtcatagtta ttgacagcgc gcctaacctg ggtatcggca cgattaatgt
     5821 cgtatgtgct gctgatgtgc tgattgttcc cacgcctgct gagttgtttg actacacctc
     5881 cgcactgcag tttttcgata tgcttcgtga tctgctcaag aacgttgatc ttaaagggtt
     5941 cgagcctgat gtacgtattt tgcttaccaa atacagcaat agtaatggct ctcagtcccc
     6001 gtggatggag gagcaaattc gggatgcctg gggaagcatg gttctaaaaa atgttgtacg
     6061 tgaaacggat gaagttggta aaggtcagat ccggatgaga actgtttttg aacaggccat
     6121 tgatcaacgc agttcaactg gtgcctggag aaatgctctt tctatttggg aacctgtctg
     6181 caatgaaatt ttcgatcgtc tgattaaacc acgctgggag attagataat gaagcgtgcg
     6241 cctgttattc caaaacatac gctcaatact caaccggttg aagatacttc gttatcgaca
     6301 ccagctgccc cgatggtgga ttcgttaatt gcgcgcgtag gagtaatggc tcgcggtaat
     6361 gccattactt tgcctgtatg tggtcgggat gtgaagttta ctcttgaagt gctccggggt
     6421 gatagtgttg agaaaacctc tcgggtatgg tcaggtaatg aacgtgacca ggagctgctt
     6481 actgaggacg cactggatga tctcatccct tcttttctac tgactggtca acagacaccg
     6541 gcgttcggtc gaagagtatc tggtgtcata gaaattgccg atgggagtcg ccgtcgtaaa
     6601 gctgctgcac ttaccgaaag tgattatcgt gttctggttg gcgagctgga tgatgagcag
     6661 atggctgcat tatccagatt gggtaacgat tatcgcccaa caagtgctta tgaacgtggt
     6721 cagcgttatg caagccgatt gcagaatgaa tttgctggaa atatttctgc gctggctgat
     6781 gcggaaaata tttcacgtaa gattattacc cgctgtatca acaccgccaa attgcctaaa
     6841 tcagttgttg ctcttttttc tcaccccggt gaactatctg cccggtcagg tgatgcactt
     6901 caaaaagcct ttacagataa agaggaatta cttaagcagc aggcatctaa ccttcatgag
     6961 cagaaaaaag ctggggtgat atttgaagct gaagaagtta tcactctttt aacttctgtg
     7021 cttaaaacgt catctgcatc aagaactagt ttaagctcac gacatcagtt tgctcctgga
     7081 gcgacagtat tgtataaggg cgataaaatg gtgcttaacc tggacaggtc acgtgttcca
     7141 actgagtgta tagagaaaat tgaggccatt cttaaggaac ttgaaaagcc agcaccctga
     7201 tgcgaccacg ttttagtcta cgtttatctg tctttactta atgtcctttg ttacaggcca
     7261 gaaagcataa ctggcctgaa tattctctct gggcccactg ttccacttgt atcgtcggtc
     7321 tgataatcag actgggacca cggtcccact cgtatcgtcg gtctgattat tagtctggga
     7381 ccacggtccc actcgtatcg tcggtctgat tattagtctg ggaccacggt cccactcgta
     7441 tcgtcggtct gataatcaga ctgggaccac ggtcccactc gtatcgtcgg tctgattatt
     7501 agtctgggac catggtccca ctcgtatcgt cggtctgatt attagtctgg gaccacggtc
     7561 ccactcgtat cgtcggtctg attattagtc tggaaccacg gtcccactcg tatcgtcggt
     7621 ctgattatta gtctgggacc acggtcccac tcgtatcgtc ggtctgatta ttagtctggg
     7681 accacgatcc cactcgtgtt gtcggtctga ttatcggtct gggaccacgg tcccacttgt
     7741 attgtcgatc agactatcag cgtgagacta cgattccatc aatgcctgtc aagggcaagt
     7801 attgacatgt cgtcgtaacc tgtagaacgg agtaacctcg gtgtgcggtt gtatgcctgc
     7861 tgtggattgc tgctgtgtcc tgcttatcca caacattttg cgcacggtta tgtggacaaa
     7921 atacctggtt acccaggccg tgccggcacg ttaaccgggc tgcatccgat gcaagtgtgt
     7981 cgctgtcgac gcaagcggcc gca
//
