LOCUS       sequence-465744-        8004 bp    DNA     circular SYN 15-APR-2025
DEFINITION  pMOBC360_StandVC: Standard Vector (Chloramphenicol 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 8004)
  TITLE     DNA assembly using methylases via targeted inhibition of type IIS 
            endonuclease activity and methylation protection by a 
            CRISPR/dCas9-based system
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8004)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: POC1552
FEATURES             Location/Qualifiers
     source          1..8004
                     /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        1834..1936
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1937..2596
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2003..2022)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      3411..3630
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3721..4476
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4479..4729
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             5055..6230
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             6230..7201
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    7274..7747
                     /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 ccgccttcag cggccgcacg aataaatacc
     1741 tgtgacggaa gatcacttcg cagaataaat aaatcctggt gtccctgttg ataccgggaa
     1801 gccctgggcc aacttttggc gaaaatgaga cgttgatcgg cacgtaagag gttccaactt
     1861 tcaccataat gaaataagat cactaccggg cgtatttttt gagttatcga gattttcagg
     1921 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca
     1981 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca
     2041 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt
     2101 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg agttccgtat
     2161 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt
     2221 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
     2281 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
     2341 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag
     2401 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
     2461 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
     2521 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
     2581 gcagggcggg gcgtaagagc tggagcggcg taaccgtcgc acaggaagga cagagaaagc
     2641 gcggatctgg gaagtgacgg acagaacggt caggacctgg attggggagg cggttgccgc
     2701 cgctgctgct gacggtgtga cgttctctgt tccggtcaca ccacatacgt tccgccattc
     2761 ctatgcgatg cacatgctgt atgccggtat accgctgaaa gttctgcaaa gcctgatggg
     2821 acataagtcc atcagttcaa cggaagtcta cacgaaggtt tttgcgctgg atgtggctgc
     2881 ccggctccgg gtgcagtttg cgatgccgga gtctgatgcg gttgcgatgc tgaaacaatt
     2941 atcctgagaa taaatgcctt ggcctttata tggaaatgtg gaactgagtg gatatgctgt
     3001 ttttgtctgt taaacagaga agctggctgt tatccactga gaagcgaacg aaacagtcgg
     3061 gaaaatctcc cattatcgta gagatccgca ttattaatct caggagcctg tgtagcgttt
     3121 ataggaagta gtgttctgtc atgatgcctg caagcggtaa cgaaaacgat ttgaatatgc
     3181 cttcaggaac aatagaaatc ttcgtgcggt gttacgttga agtggagcgg attatgtcag
     3241 caatggacag aacaacctaa tgaacacaga accatgatgt ggtctgtcct tttacagcca
     3301 gtagtgctcg ccgcagtcga gcgacagggc gaagccctcg agtgagcgag gaagcaccag
     3361 ggaacagcac ttatatattc tgcttacaca cgatgcctga aaaaacttcc cttggggtta
     3421 tccacttatc cacggggata tttttataat tatttttttt atagttttta gatcttcttt
     3481 tttagagcgc cttgtaggcc tttatccatg ctggttctag agaaggtgtt gtgacaaatt
     3541 gccctttcag tgtgacaaat caccctcaaa tgacagtcct gtctgtgaca aattgccctt
     3601 aaccctgtga caaattgccc tcagaagaag ctgttttttc acaaagttat ccctgcttat
     3661 tgactctttt ttatttagtg tgacaatcta aaaacttgtc acacttcaca tggatctgtc
     3721 atggcggaaa cagcggttat caatcacaag aaacgtaaaa atagcccgcg aatcgtccag
     3781 tcaaacgacc tcactgaggc ggcatatagt ctctcccggg atcaaaaacg tatgctgtat
     3841 ctgttcgttg accagatcag aaaatctgat ggcaccctac aggaacatga cggtatctgc
     3901 gagatccatg ttgctaaata tgctgaaata ttcggattga cctctgcgga agccagtaag
     3961 gatatacggc aggcattgaa gagtttcgcg gggaaggaag tggtttttta tcgccctgaa
     4021 gaggatgccg gcgatgaaaa aggctatgaa tcttttcctt ggtttatcaa acgtgcgcac
     4081 agtccatcca gagggcttta cagtgtacat atcaacccat atctcattcc cttctttatc
     4141 gggttacaga accggtttac gcagtttcgg cttagtgaaa caaaagaaat caccaatccg
     4201 tatgccatgc gtttatacga atccctgtgt cagtatcgta agccggatgg ctcaggcatc
     4261 gtgtctctga aaatcgactg gatcatagag cgttaccagc tgcctcaaag ttaccagcgt
     4321 atgcctgact tccgccgccg cttcctgcag gtctgtgtta atgagatcaa cagcagaact
     4381 ccaatgcgcc tctcatacat tgagaaaaag aaaggccgcc agacgactca tatcgtattt
     4441 tccttccgcg atatcacttc catgacgaca ggatagtctg agggttatct gtcacagatt
     4501 tgagggtggt tcgtcacatt tgttctgacc tactgagggt aatttgtcac agttttgctg
     4561 tttccttcag cctgcatgga ttttctcata ctttttgaac tgtaattttt aaggaagcca
     4621 aatttgaggg cagtttgtca cagttgattt ccttctcttt cccttcgtca tgtgacctga
     4681 tatcgggggt tagttcgtca tcattgatga gggttgatta tcacagttta ttactctgaa
     4741 ttggctatcc gcgtgtgtac ctctacctgg agtttttccc acggtggata tttcttcttg
     4801 cgctgagcgt aagagctatc tgacagaaca gttcttcttt gcttcctcgc cagttcgctc
     4861 gctatgctcg gttacacggc tgcggcgagc gctagtgata ataagtgact gaggtatgtg
     4921 gtgttcttat ctccttttgt agtgttgctc ttattttaaa caactttgcg gttttttgat
     4981 gactttgcga ttttgttgtt gctttgcagt aaattgcaag atttaataaa aaaacgcaaa
     5041 gcaatgatta aaggatgttc agaatgaaac tcatggaaac acttaaccag tgcataaacg
     5101 ctggtcatga aatgacgaag gctatcgcca ttgcacagtt taatgatgac agcccggaag
     5161 cgaggaaaat aacccggcgc tggagaatag gtgaagcagc ggatttagtt ggggtttctt
     5221 ctcaggctat cagagatgcc gagaaagcag ggcgactacc gcacccggat atggaaattc
     5281 gaggacgggt tgagcaacgt gttggttata caattgaaca aattaatcat atgcgtgatg
     5341 tgtttggtac gcgattgcga cgtgctgagg acgtatttcc accggtgatc ggggttgctg
     5401 cccataaagg tggcgtttac aaaacctcag tttctgttca tcttgctcag gatctggctc
     5461 tgaaggggct acgtgttttg ctcgtggaag gtaacgaccc ccagggaaca gcctcaatgt
     5521 atcacggatg ggtaccagat cttcatattc atgcagaaga tactctcctg cctttctatc
     5581 ttggggaaaa ggacgatgtc acttatgcaa taaagcccac ttgctggccg gggcttgaca
     5641 ttattccttc ctgtctggct ctgcaccgta ttgaaactga gttaatgggc aaatttgatg
     5701 aaggtaaact gcccaccgat ccacacctga tgctccgact ggccattgaa actgttgctc
     5761 atgactatga tgtcatagtt attgacagcg cgcctaacct gggtatcggc acgattaatg
     5821 tcgtatgtgc tgctgatgtg ctgattgttc ccacgcctgc tgagttgttt gactacacct
     5881 ccgcactgca gtttttcgat atgcttcgtg atctgctcaa gaacgttgat cttaaagggt
     5941 tcgagcctga tgtacgtatt ttgcttacca aatacagcaa tagtaatggc tctcagtccc
     6001 cgtggatgga ggagcaaatt cgggatgcct ggggaagcat ggttctaaaa aatgttgtac
     6061 gtgaaacgga tgaagttggt aaaggtcaga tccggatgag aactgttttt gaacaggcca
     6121 ttgatcaacg cagttcaact ggtgcctgga gaaatgctct ttctatttgg gaacctgtct
     6181 gcaatgaaat tttcgatcgt ctgattaaac cacgctggga gattagataa tgaagcgtgc
     6241 gcctgttatt ccaaaacata cgctcaatac tcaaccggtt gaagatactt cgttatcgac
     6301 accagctgcc ccgatggtgg attcgttaat tgcgcgcgta ggagtaatgg ctcgcggtaa
     6361 tgccattact ttgcctgtat gtggtcggga tgtgaagttt actcttgaag tgctccgggg
     6421 tgatagtgtt gagaaaacct ctcgggtatg gtcaggtaat gaacgtgacc aggagctgct
     6481 tactgaggac gcactggatg atctcatccc ttcttttcta ctgactggtc aacagacacc
     6541 ggcgttcggt cgaagagtat ctggtgtcat agaaattgcc gatgggagtc gccgtcgtaa
     6601 agctgctgca cttaccgaaa gtgattatcg tgttctggtt ggcgagctgg atgatgagca
     6661 gatggctgca ttatccagat tgggtaacga ttatcgccca acaagtgctt atgaacgtgg
     6721 tcagcgttat gcaagccgat tgcagaatga atttgctgga aatatttctg cgctggctga
     6781 tgcggaaaat atttcacgta agattattac ccgctgtatc aacaccgcca aattgcctaa
     6841 atcagttgtt gctctttttt ctcaccccgg tgaactatct gcccggtcag gtgatgcact
     6901 tcaaaaagcc tttacagata aagaggaatt acttaagcag caggcatcta accttcatga
     6961 gcagaaaaaa gctggggtga tatttgaagc tgaagaagtt atcactcttt taacttctgt
     7021 gcttaaaacg tcatctgcat caagaactag tttaagctca cgacatcagt ttgctcctgg
     7081 agcgacagta ttgtataagg gcgataaaat ggtgcttaac ctggacaggt cacgtgttcc
     7141 aactgagtgt atagagaaaa ttgaggccat tcttaaggaa cttgaaaagc cagcaccctg
     7201 atgcgaccac gttttagtct acgtttatct gtctttactt aatgtccttt gttacaggcc
     7261 agaaagcata actggcctga atattctctc tgggcccact gttccacttg tatcgtcggt
     7321 ctgataatca gactgggacc acggtcccac tcgtatcgtc ggtctgatta ttagtctggg
     7381 accacggtcc cactcgtatc gtcggtctga ttattagtct gggaccacgg tcccactcgt
     7441 atcgtcggtc tgataatcag actgggacca cggtcccact cgtatcgtcg gtctgattat
     7501 tagtctggga ccatggtccc actcgtatcg tcggtctgat tattagtctg ggaccacggt
     7561 cccactcgta tcgtcggtct gattattagt ctggaaccac ggtcccactc gtatcgtcgg
     7621 tctgattatt agtctgggac cacggtccca ctcgtatcgt cggtctgatt attagtctgg
     7681 gaccacgatc ccactcgtgt tgtcggtctg attatcggtc tgggaccacg gtcccacttg
     7741 tattgtcgat cagactatca gcgtgagact acgattccat caatgcctgt caagggcaag
     7801 tattgacatg tcgtcgtaac ctgtagaacg gagtaacctc ggtgtgcggt tgtatgcctg
     7861 ctgtggattg ctgctgtgtc ctgcttatcc acaacatttt gcgcacggtt atgtggacaa
     7921 aatacctggt tacccaggcc gtgccggcac gttaaccggg ctgcatccga tgcaagtgtg
     7981 tcgctgtcga cgcaagcggc cgca
//
