LOCUS       40924_29876        6419 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pMB18424, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6419)
  AUTHORS   Batista MB.
  TITLE     pMB18424, a new tetA-sacB vector for counterselection in bacteria
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6419)
  AUTHORS   Batista MB.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-FEB-2018) Molecular Microbiology, John Innes Centre, 
            Colney Laney, Norwich, Norfolk NR4 7PH, United Kingdom
REFERENCE   3  (bases 1 to 6419)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6419)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (14-FEB-2018) Molecular Microbiology, John Innes Centre, Colney 
            Laney, Norwich, Norfolk NR4 7PH, United Kingdom"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     709..732
                     /label=M13 F(-47)
                     /note="M13 F(-47)"
     primer_bind     736..752
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        762..780
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    complement(789..896)
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     promoter        complement(909..927)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(948..964)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(972..988)
                     /label=lac operator
                     /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)."
     promoter        complement(996..1026)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1041..1062)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(1224..2411)
                     /codon_start=1
                     /label=TcR
                     /note="tetracycline efflux protein"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     promoter        complement(2459..2487)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     promoter        2519..2964
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             2965..4383
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     oriT            complement(4705..4814)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     promoter        complement(5049..5139)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     promoter        complement(5333..5423)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      5783..6371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
       61 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      121 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      181 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      241 aatgtgaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
      301 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
      361 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtcaa gcactaaatc
      421 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
      481 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
      541 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
      601 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
      661 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
      721 cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg
      781 gcgaattggg taccgggccc cccctcgagg tcgacggtat cgataagctt gatatcgaat
      841 tcctgcagcc cgggggatcc actagttcta gagcggccgc caccgcggtg gagctccagc
      901 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
      961 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     1021 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     1081 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1141 gggagaggcg gtttgcgtat tgggcgctct tccaattctt ggagtggtga atccgttagc
     1201 gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac
     1261 gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg
     1321 ctcgccgagg cggcataaat cgccgtgacg atcagcggtc cagtgatcga agttaggctg
     1381 gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac
     1441 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg
     1501 aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc
     1561 atgccggcga taatggcctg cttctcgccg aaacgcttgg tggcgggacc agtgacgaag
     1621 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     1681 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     1741 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     1801 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcggcgctc tcccttatgc
     1861 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     1921 aggaatggtg catgtaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     1981 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     2041 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     2101 atgcgtccgg cgtagagaat ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata
     2161 gtggctccaa gtagcgaagc gagcaggact gggcggcggc caaagcggtc ggacagtgct
     2221 ccgagaacgg gtgcgcatag aaattgcatc aacgcatata gcgctagcag cacgccatag
     2281 tgactggcga tgctgtcgga atggacgata tcccgcaaga ggcccggcag taccggcata
     2341 accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg
     2401 ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat
     2461 taaagctaat cgatgataag ctgtcaaaca tgaggaggat cgatcctttt taacccatca
     2521 catatacctg ccgttcacta ttatttagtg aaatgagata ttatgatatt ttctgaattg
     2581 tgattaaaaa ggcaacttta tgcccatgca acagaaacta taaaaaatac agagaatgaa
     2641 aagaaacaga tagatttttt agttctttag gcccgtagtc tgcaaatcct tttatgattt
     2701 tctatcaaac aaaagaggaa aatagaccag ttgcaatcca aacgagagtc taatagaatg
     2761 aggtcgaaaa gtaaatcgcg cgggtttgtt actgataaag caggcaagac ctaaaatgtg
     2821 taaagggcaa agtgtatact ttggcgtcac cccttacata ttttaggtct ttttttattg
     2881 tgcgtaacta acttgccatc ttcaaacagg agggctggaa gaagcagacc gctaacacag
     2941 tacataaaaa aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta
     3001 ttaaccttta ctaccgcact gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg
     3061 aaccaaaagc catataagga aacatacggc atttcccata ttacacgcca tgatatgctg
     3121 caaatccctg aacagcaaaa aaatgaaaaa tatcaagttt ctgaatttga ttcgtccaca
     3181 attaaaaata tctcttctgc aaaaggcctg gacgtttggg acagctggcc attacaaaac
     3241 gctgacggca ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt agccggagat
     3301 cctaaaaatg cggatgacac atcgatttac atgttctatc aaaaagtcgg cgaaacttct
     3361 attgacagct ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat
     3421 gattctatcc taaaagacca aacacaagaa tggtcaggtt cagccacatt tacatctgac
     3481 ggaaaaatcc gtttattcta cactgatttc tccggtaaac attacggcaa acaaacactg
     3541 acaactgcac aagttaacgt atcagcatca gacagctctt tgaacatcaa cggtgtagag
     3601 gattataaat caatctttga cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc
     3661 gatgaaggca actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa
     3721 gataaaggcc acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa
     3781 ggcgaagaat ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa
     3841 gaaagtcaaa aacttctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct
     3901 ctcggtatga ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt
     3961 gcatctaaca cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa
     4021 tggtacctgt tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac
     4081 gatatttaca tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac
     4141 aaaactggcc ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca
     4201 cacttcgctg tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac
     4261 agaggattct acgcagacaa acaatcaacg tttgcgccga gcttcctgct gaacatcaaa
     4321 ggcaagaaaa catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac
     4381 aaataaaaac gcaaaagaaa atgccgatgg gtaccgagcg cgagatttcg attccaccgc
     4441 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccctcg cggacgtgct
     4501 catagtccac gacgcccgtg attttgtagc cctggccgac ggccagcagg taggccgaca
     4561 ggctcatgcc ggccgccgcc gccttttcct caatcgctct tcgttcgtct ggaaggcagt
     4621 acaccttgat aggtgggctg cccttcctgg ttggcttggt ttcatcagcc atccgcttgc
     4681 cctcatctgt tacgccggcg gtagccggcc agcctcgcag agcaggattc ccgttgagca
     4741 ccgccaggtg cgaataaggg acagtgaaga aggaacaccc gctcgcgggt gggcctactt
     4801 cacctatcct gcccggctga cgccgttgga tacaccaagg aaagtctaca cgaacccttt
     4861 ggcaaaatcc tgtatatcgt gcgaaaaagg atggatatac cgaaaaaatc gctataatga
     4921 ccccgaagca gggttatgca gcggaaaagc gctgcttccc tgctgttttg tggaatatct
     4981 accgactgga aacaggcaaa tgcaggaaat tactgaactg aggggacagg cgagagacga
     5041 tgccaaagag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt
     5101 tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag
     5161 ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga
     5221 tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttactg
     5281 atttagtgta tgatggtgtt tttgaggtgc tccagtggct tctgtttcta tcagctcctg
     5341 aaaatctcga taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt
     5401 tggaacctct tacgtgccga tcaacgtctc attttcgcca aaagttggcc cagggcttcc
     5461 cggtatcaac agggacacca ggatttattt attctgcgaa gtgatcttcc gtcacaggta
     5521 tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt actgatttag tgtatgatgg
     5581 tgtttttgag gtgctccagt ggcttctgtt tctatcaggg ctggatgatc ctccagcgcg
     5641 gggatctcat gctggagttc ttcgcccacc ccaaaaggat ctaggtgaag atcctttttg
     5701 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5761 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5821 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5881 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5941 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6001 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6061 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6121 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6181 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6241 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6301 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6361 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcct
//
