LOCUS Exported 8068 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION POC12404, MetClo assembly vector with F replication origin and ampicillin resistance for BsaI-based MetClo, adaptor sequence type a-b. ACCESSION . VERSION . KEYWORDS pMXBP_aFb SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8068) 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 8068) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8068 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 223..811 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 712..731 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 965..982 /label=L4440 /note="L4440 vector, forward primer" protein_bind 1099..1120 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 1135..1165 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1173..1189 /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 1178..1200 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 1197..1213 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 1197..1213 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 1209..1532 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS DAA" misc_feature 1226..1282 /label=MCS /note="pUC18/19 multiple cloning site" primer_bind complement(1283..1300) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1283..1299) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1292..1314) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(1508..1527) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 1695..1799 /gene="bla" /label=AmpR promoter CDS 1800..2660 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(2018..2037) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3475..3694 /label=ori2 /note="secondary origin of replication for the bacterial F plasmid; also known as oriS" CDS 3785..4540 /codon_start=1 /gene="repE" /product="replication initiation protein for the bacterial F plasmid" /label=repE /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM RLSYIEKKKGRQTTHIVFSFRDITSMTTG" misc_feature 4543..4793 /gene="incC" /label=incC /note="incompatibility region of the bacterial F plasmid" CDS 5119..6294 /codon_start=1 /gene="sopA" /product="partitioning protein for the bacterial F plasmid" /label=sopA /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR" CDS 6294..7265 /codon_start=1 /gene="sopB" /product="partitioning protein for the bacterial F plasmid" /label=sopB /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP" misc_feature 7338..7811 /gene="sopC" /label=sopC /note="centromere-like partitioning region of the bacterial F plasmid" ORIGIN 1 cactctgtga cttggtctca aggtggagtg agaccggcct gcaggtgtga gtttgtcaga 61 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 121 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 181 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 241 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 301 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 361 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 421 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 481 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 541 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 601 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 661 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 721 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 781 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 841 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 901 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 961 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 1021 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 1081 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 1141 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 1201 aaacagctat gaccatgatt acgccaagct tgcatgcctg caggtcgact ctagaggatc 1261 cccgggtacc gagctcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 1321 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 1381 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 1441 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 1501 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 1561 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 1621 caagtaccac ctggaacgcc aaaggtctca cgcttgcctg agaccaagtc acgaagtgtg 1681 cggccgcctt cgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 1741 atccgctcat gagacaataa ccttgataaa tgcttcaata atattgaaaa aggaagagta 1801 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 1861 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 1921 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 1981 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 2041 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 2101 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 2161 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 2221 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 2281 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 2341 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 2401 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 2461 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttctc 2521 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 2581 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 2641 cactgattaa gcattggtaa gagctggagc ggcgtaaccg tcgcacagga aggacagaga 2701 aagcgcggat ctgggaagtg acggacagaa cggtcaggac ctggattggg gaggcggttg 2761 ccgccgctgc tgctgacggt gtgacgttct ctgttccggt cacaccacat acgttccgcc 2821 attcctatgc gatgcacatg ctgtatgccg gtataccgct gaaagttctg caaagcctga 2881 tgggacataa gtccatcagt tcaacggaag tctacacgaa ggtttttgcg ctggatgtgg 2941 ctgcccggct ccgggtgcag tttgcgatgc cggagtctga tgcggttgcg atgctgaaac 3001 aattatcctg agaataaatg ccttggcctt tatatggaaa tgtggaactg agtggatatg 3061 ctgtttttgt ctgttaaaca gagaagctgg ctgttatcca ctgagaagcg aacgaaacag 3121 tcgggaaaat ctcccattat cgtagagatc cgcattatta atctcaggag cctgtgtagc 3181 gtttatagga agtagtgttc tgtcatgatg cctgcaagcg gtaacgaaaa cgatttgaat 3241 atgccttcag gaacaataga aatcttcgtg cggtgttacg ttgaagtgga gcggattatg 3301 tcagcaatgg acagaacaac ctaatgaaca cagaaccatg atgtggtctg tccttttaca 3361 gccagtagtg ctcgccgcag tcgagcgaca gggcgaagcc ctcgagtgag cgaggaagca 3421 ccagggaaca gcacttatat attctgctta cacacgatgc ctgaaaaaac ttcccttggg 3481 gttatccact tatccacggg gatattttta taattatttt ttttatagtt tttagatctt 3541 cttttttaga gcgccttgta ggcctttatc catgctggtt ctagagaagg tgttgtgaca 3601 aattgccctt tcagtgtgac aaatcaccct caaatgacag tcctgtctgt gacaaattgc 3661 ccttaaccct gtgacaaatt gccctcagaa gaagctgttt tttcacaaag ttatccctgc 3721 ttattgactc ttttttattt agtgtgacaa tctaaaaact tgtcacactt cacatggatc 3781 tgtcatggcg gaaacagcgg ttatcaatca caagaaacgt aaaaatagcc cgcgaatcgt 3841 ccagtcaaac gacctcactg aggcggcata tagtctctcc cgggatcaaa aacgtatgct 3901 gtatctgttc gttgaccaga tcagaaaatc tgatggcacc ctacaggaac atgacggtat 3961 ctgcgagatc catgttgcta aatatgctga aatattcgga ttgacctctg cggaagccag 4021 taaggatata cggcaggcat tgaagagttt cgcggggaag gaagtggttt tttatcgccc 4081 tgaagaggat gccggcgatg aaaaaggcta tgaatctttt ccttggttta tcaaacgtgc 4141 gcacagtcca tccagagggc tttacagtgt acatatcaac ccatatctca ttcccttctt 4201 tatcgggtta cagaaccggt ttacgcagtt tcggcttagt gaaacaaaag aaatcaccaa 4261 tccgtatgcc atgcgtttat acgaatccct gtgtcagtat cgtaagccgg atggctcagg 4321 catcgtgtct ctgaaaatcg actggatcat agagcgttac cagctgcctc aaagttacca 4381 gcgtatgcct gacttccgcc gccgcttcct gcaggtctgt gttaatgaga tcaacagcag 4441 aactccaatg cgcctctcat acattgagaa aaagaaaggc cgccagacga ctcatatcgt 4501 attttccttc cgcgatatca cttccatgac gacaggatag tctgagggtt atctgtcaca 4561 gatttgaggg tggttcgtca catttgttct gacctactga gggtaatttg tcacagtttt 4621 gctgtttcct tcagcctgca tggattttct catacttttt gaactgtaat ttttaaggaa 4681 gccaaatttg agggcagttt gtcacagttg atttccttct ctttcccttc gtcatgtgac 4741 ctgatatcgg gggttagttc gtcatcattg atgagggttg attatcacag tttattactc 4801 tgaattggct atccgcgtgt gtacctctac ctggagtttt tcccacggtg gatatttctt 4861 cttgcgctga gcgtaagagc tatctgacag aacagttctt ctttgcttcc tcgccagttc 4921 gctcgctatg ctcggttaca cggctgcggc gagcgctagt gataataagt gactgaggta 4981 tgtggtgttc ttatctcctt ttgtagtgtt gctcttattt taaacaactt tgcggttttt 5041 tgatgacttt gcgattttgt tgttgctttg cagtaaattg caagatttaa taaaaaaacg 5101 caaagcaatg attaaaggat gttcagaatg aaactcatgg aaacacttaa ccagtgcata 5161 aacgctggtc atgaaatgac gaaggctatc gccattgcac agtttaatga tgacagcccg 5221 gaagcgagga aaataacccg gcgctggaga ataggtgaag cagcggattt agttggggtt 5281 tcttctcagg ctatcagaga tgccgagaaa gcagggcgac taccgcaccc ggatatggaa 5341 attcgaggac gggttgagca acgtgttggt tatacaattg aacaaattaa tcatatgcgt 5401 gatgtgtttg gtacgcgatt gcgacgtgct gaggacgtat ttccaccggt gatcggggtt 5461 gctgcccata aaggtggcgt ttacaaaacc tcagtttctg ttcatcttgc tcaggatctg 5521 gctctgaagg ggctacgtgt tttgctcgtg gaaggtaacg acccccaggg aacagcctca 5581 atgtatcacg gatgggtacc agatcttcat attcatgcag aagatactct cctgcctttc 5641 tatcttgggg aaaaggacga tgtcacttat gcaataaagc ccacttgctg gccggggctt 5701 gacattattc cttcctgtct ggctctgcac cgtattgaaa ctgagttaat gggcaaattt 5761 gatgaaggta aactgcccac cgatccacac ctgatgctcc gactggccat tgaaactgtt 5821 gctcatgact atgatgtcat agttattgac agcgcgccta acctgggtat cggcacgatt 5881 aatgtcgtat gtgctgctga tgtgctgatt gttcccacgc ctgctgagtt gtttgactac 5941 acctccgcac tgcagttttt cgatatgctt cgtgatctgc tcaagaacgt tgatcttaaa 6001 gggttcgagc ctgatgtacg tattttgctt accaaataca gcaatagtaa tggctctcag 6061 tccccgtgga tggaggagca aattcgggat gcctggggaa gcatggttct aaaaaatgtt 6121 gtacgtgaaa cggatgaagt tggtaaaggt cagatccgga tgagaactgt ttttgaacag 6181 gccattgatc aacgcagttc aactggtgcc tggagaaatg ctctttctat ttgggaacct 6241 gtctgcaatg aaattttcga tcgtctgatt aaaccacgct gggagattag ataatgaagc 6301 gtgcgcctgt tattccaaaa catacgctca atactcaacc ggttgaagat acttcgttat 6361 cgacaccagc tgccccgatg gtggattcgt taattgcgcg cgtaggagta atggctcgcg 6421 gtaatgccat tactttgcct gtatgtggtc gggatgtgaa gtttactctt gaagtgctcc 6481 ggggtgatag tgttgagaaa acctctcggg tatggtcagg taatgaacgt gaccaggagc 6541 tgcttactga ggacgcactg gatgatctca tcccttcttt tctactgact ggtcaacaga 6601 caccggcgtt cggtcgaaga gtatctggtg tcatagaaat tgccgatggg agtcgccgtc 6661 gtaaagctgc tgcacttacc gaaagtgatt atcgtgttct ggttggcgag ctggatgatg 6721 agcagatggc tgcattatcc agattgggta acgattatcg cccaacaagt gcttatgaac 6781 gtggtcagcg ttatgcaagc cgattgcaga atgaatttgc tggaaatatt tctgcgctgg 6841 ctgatgcgga aaatatttca cgtaagatta ttacccgctg tatcaacacc gccaaattgc 6901 ctaaatcagt tgttgctctt ttttctcacc ccggtgaact atctgcccgg tcaggtgatg 6961 cacttcaaaa agcctttaca gataaagagg aattacttaa gcagcaggca tctaaccttc 7021 atgagcagaa aaaagctggg gtgatatttg aagctgaaga agttatcact cttttaactt 7081 ctgtgcttaa aacgtcatct gcatcaagaa ctagtttaag ctcacgacat cagtttgctc 7141 ctggagcgac agtattgtat aagggcgata aaatggtgct taacctggac aggtcacgtg 7201 ttccaactga gtgtatagag aaaattgagg ccattcttaa ggaacttgaa aagccagcac 7261 cctgatgcga ccacgtttta gtctacgttt atctgtcttt acttaatgtc ctttgttaca 7321 ggccagaaag cataactggc ctgaatattc tctctgggcc cactgttcca cttgtatcgt 7381 cggtctgata atcagactgg gaccacggtc ccactcgtat cgtcggtctg attattagtc 7441 tgggaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc acggtcccac 7501 tcgtatcgtc ggtctgataa tcagactggg accacggtcc cactcgtatc gtcggtctga 7561 ttattagtct gggaccatgg tcccactcgt atcgtcggtc tgattattag tctgggacca 7621 cggtcccact cgtatcgtcg gtctgattat tagtctggaa ccacggtccc actcgtatcg 7681 tcggtctgat tattagtctg ggaccacggt cccactcgta tcgtcggtct gattattagt 7741 ctgggaccac gatcccactc gtgttgtcgg tctgattatc ggtctgggac cacggtccca 7801 cttgtattgt cgatcagact atcagcgtga gactacgatt ccatcaatgc ctgtcaaggg 7861 caagtattga catgtcgtcg taacctgtag aacggagtaa cctcggtgtg cggttgtatg 7921 cctgctgtgg attgctgctg tgtcctgctt atccacaaca ttttgcgcac ggttatgtgg 7981 acaaaatacc tggttaccca ggccgtgccg gcacgttaac cgggctgcat ccgatgcaag 8041 tgtgtcgctg tcgacgcaag cggccgca //