LOCUS       V016399                 7997 bp    DNA     circular SYN 28-JUL-2021
DEFINITION  Exported.
ACCESSION   V016399
VERSION     V016399
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7997)
  AUTHORS   Lau MSH., Sheng L, Zhang Y, Minton NP.
  TITLE     Development of a Suite of Tools for Genome Editing in
            Parageobacillus thermoglucosidasius and Their Use to Identify the
            Potential of a Native Plasmid in the Generation of Stable Engineered
            Strains
  JOURNAL   ACS Synth Biol 10 (7), 1739-1749 (2021)
   PUBMED   34197093
REFERENCE   2  (bases 1 to 7997)
  AUTHORS   Lau MSH., Sheng L, Zhang Y, Minton NP.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-MAY-2021) Biodiscovery Institute, School of Life
            Sciences, University of Nottingham, Science Road, University Park,
            Nottingham, Nottinghamshire NG7 2RD, United Kingdom
REFERENCE   3  (bases 1 to 7997)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
            Biol"; date: "2021"; volume: "10"; issue: "7"; pages: "1739-1749"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (10-MAY-2021) Biodiscovery Institute, School of Life Sciences,
            University of Nottingham, Science Road, University Park, Nottingham,
            Nottinghamshire NG7 2RD, United Kingdom"
FEATURES             Location/Qualifiers
     source          1..7997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(12..4175)
                     /gene="cas9-2"
                     /label="CRISPR-associated endonuclease Cas9 2"
                     /note="CRISPR-associated endonuclease Cas9 2 from
                     Streptococcus thermophilus (strain ATCC BAA-491 / LMD-9).
                     Accession#: Q03JI6"
     regulatory      complement(4176..4360)
                     /label="lactate dehydrogenase promoter and RBS"
                     /note="lactate dehydrogenase promoter and RBS"
                     /regulatory_class="promoter"
     regulatory      4367..4457
                     /label="glyceraldehyde 3-phosphate dehydrogenase promoter"
                     /note="glyceraldehyde 3-phosphate dehydrogenase promoter"
                     /regulatory_class="promoter"
     terminator      4595..4681
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4773..4800
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     primer_bind     complement(4810..4826)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     CDS             5262..6263
                     /label="repB"
                     /note="RepB replication protein"
     CDS             6420..7190
                     /codon_start=1
                     /transl_table=11
                     /product="kanamycin nucleotidyltransferase"
                     /label="kanamycin nucleotidyltransferase"
                     /protein_id="QXV87314.1"
                     /translation="MRIVNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGR
                     QTDGPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFYSEEILLDYASQVESDWPLTHG
                     QFFSILPIYDSGGYLEKVYQTAKSVEAQKFHDAICALIVEELFEYAGKWRNIRVQGPTT
                     FLPSLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSD
                     SEKLLESLENFWNGIQEWTERHGYIVDVSKRIPF"
     rep_origin      7198..7781
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
ORIGIN
        1 cctgcaggtt aaccctctcc tagtttggca aggtctattc gtgtttcata gaggcctgta
       61 acagattgat gaataagtgt ggcatctttt aatagggatg atggggtata gtctctatac
      121 cttggaattt taacacctaa aaattcaaaa tcagcagcac ttccacgaga ggttaattca
      181 aatagcccct ttctttcact tccggtaggt cctataaaac tactacagag ttcatctata
      241 ctatgatttt gccaagattg aaaggcagag tttaagagtt taccattctt tttagctcca
      301 acataattct cattaaactc aagaatgtag taaaataatt cttcaaactc ttttttatgg
      361 ttctcaacat attttctatg attctcatta attgtgttac ttattctctt agcatgataa
      421 agtaatttca caaacttctg tgaaagaaaa atctgatttc ctttgtgaat ctctcccctc
      481 ttattattcg ttgacaaaat actagccaac atacgacgtg aaccatctga aagttcaaat
      541 aaactatatt taggtagttc aataattaac tcaatatctt tataaccttt ttcaagtaaa
      601 aaattaagtt tatcttttct ataattaatc ctatctaaaa tagaaatacc ttgaaattct
      661 agtacatttg ttattttttt cttagcacct ttttcaattg tccctttaac aagaacagta
      721 aaagaattag aaattccagc atacccccca tactttttgg ggtcaagata ctctttagca
      781 cctactaaat tttcattact atttggtttt ggctttgagg aaagatttgc attaaacaat
      841 ccctttggtt ttcctctatc caatccgtga ttctgttcct caactttttt cacaacattt
      901 acttgcggat aagagagtac tctccttaca gttgctaaat cagattcttt attccaaacg
      961 gattcgccgg tctcctcatt tacctcaata agtggtcttt caataactct accatcagct
     1021 aaagaaatag attttttaaa gatattcatg atatttgaat agaaatatac cttttctgta
     1081 gcggactttc tttctctaaa actattgtat tttggataat caccgtacac aaattctggc
     1141 tctagtttag ggtatttctt aagtaaagca cttgctacaa cggcattcaa gtaagcatca
     1201 tgagcatgat gaaaatcatt gatttcacga actttataaa gttcaaaatc cttacgaaat
     1261 tgagaaacta aggtagattt caaggtaata atttttactg ttcgtaccgc tctattattt
     1321 tcatcttttt tattattaaa tttctcatca agtaaacgag ctacatgttt tgttatttga
     1381 cgtgtttcaa ccaactggcg ttgaataaaa ccagctttgt cctcaggtga caatcctccc
     1441 cgttcagctt ttgtcagatt atcaaatttt cgttgagaaa ttaattttga tttcaataat
     1501 tgataccaaa atgtctttct ttttttgaca acctctaaac ttggaacatc atctgattta
     1561 ccacggttac tagcagatga aacaagtact ttattgtcaa tagaattatc tttcaaaaaa
     1621 gcttgaggaa taatatgatc aatatcataa ttacttaatc tatcaatatc taaatcatct
     1681 ccggtataca tatcttttcc attttgaaga tagtataagt aaagtcgatc attttgaagt
     1741 gcgttattgt ctattttaga aagttttgca ggaatatttt ccttaagtat cttactacct
     1801 aactctttga gagatttttc taaacgtttc aagcgttgtt gggaattaga cttaccttga
     1861 ttggtatatt gattttcacg agccatctca acaacaattg actcgggttt tcttcctccc
     1921 attactttga ccaattcatc tacaattttt atgctttgta aaataccttt tttaatcgca
     1981 ggactacctg gcaaagactt aacgacctct ttaatattac ctttatcttc gtcaccaata
     2041 atttgtgctt tctgtatctt ctttttaaaa gaaagagcat catcgtgaat aagttgcatg
     2101 aaattacggt tagaaatacc atcatcaatt aagtaatcaa gaatagtatt accagatttt
     2161 tcatctcgaa taccattaat aagcttagca gataacttac cccagccagt gtaatgtcta
     2221 cgagataact ttttcaaaac ggatttatcg aatatattct caaattttga aagacgttgt
     2281 tttatcatct ctctatcttc aaaaattgtc aaagtatgga taatttcttc gataatcgct
     2341 tcatttgaac tatcatccaa aaactcttta tcattaataa tatttaaaag atcgtgataa
     2401 gtagataaac tagaattaaa ctgtttctct atgcctttta attcaattcc atcataccca
     2461 taaattgcat gtaaatattc aataatatcc ttatcagtaa ctttcctttt atctttaaaa
     2521 taaagtctaa caatatcttt cttctgctta ctatctaaaa attgataatc tctcatactt
     2581 tcggcaataa atctaacttt tgttaattca ttatatacat taaaagtttc gtataagaga
     2641 ctatgctttg gaagtacctt ctcttctggc aaatacaaat caaaactagt cattcgatta
     2701 atgaaggcct ctgccgaaga ttctttgtca ataacgtcct caaaattcca aggtgtaatt
     2761 ttttcatttc gttttcttat tgaccaggca aaatcactat tccctctcgc aagtggacct
     2821 acataataag gaattcggaa ggttaaaatc ttctcgattc tttctttatt tttagccaag
     2881 aaaggataaa atttagcttg cttatcaaga attgctctca tttcttgaag atgaatctga
     2941 tatggtatcg aaccattgtc aaatgtacgt tgctttctca aaaaatcttc tcgatcaatt
     3001 ttttcaagaa aataatccgc accttcaaat tcagccaata gtttttttag atatacgtag
     3061 aaatcttcct gatttgtttt tccatcaata taaccagcat aaccattttt ggtgtcatct
     3121 ttaaatactt cattatacgt ttttagtgaa atatttctta tatattcctt tagtaacgct
     3181 aaatcttctt tgtgttcatt atatcgcttt atcatagcag aagagagagg tgcttctgtc
     3241 tcattatcag ttacagtcag aaaacccgat aaaagaatag catcataaag tttctttgct
     3301 ttgagaaaga catcactgta atcatctcca atataaccta acaaagtctc taaatcttca
     3361 tcatagcttt ctttggaaaa atgtaaggag gctttttcgt ctaaattaaa acatttccta
     3421 aaatcagctt gatttcctac aatcaacttt agaaactctg aaaaaatccc cgaattcttc
     3481 tccccaggga agagttttaa aatacgatct ttcttttcta atttactaat cttatcttta
     3541 acaatttcct caagttgttt actattctca agtgataaat ccgattcaaa aatagcatta
     3601 taagtgtcca aaaagtcttg aaaattcttc tgaatatcat tattttttga attaaactct
     3661 ccttcaatta agaagtgacc tctatattta atcatatgag ccaatgcaag ataaactaga
     3721 cgcaaatctg cttttttagt actatctgct aaatatttcc ttaaatgata gatagttgga
     3781 aattcatcat gataggcttt ttcttctact aagtttccaa atatcggata cttactatca
     3841 cgtttatcat caggaactaa aaacgaatcg tcaagtcttt gaaagaaagc atcatctaat
     3901 gtagccatct ctgtgctaaa aatttcctgc aaataaagga tacgattacg gcgtctagta
     3961 taacgtcttc ttgcagtacg cttcaatctt cttccttctg ctgtgattcc agagtcaaag
     4021 agtaatacac ctaacaggtt ctttttgata tactttttac tcgtatttcc taagactttc
     4081 atttttttag acggaacctt gtaattatca gttgttacag cccatccaac actattcgtt
     4141 ccaatatcaa gtccaattga gtatggctta gtcattgcat tcatcctccc tcaatataat
     4201 gcgaacactt tcatataaat attttacgcg aaaggcgcac cgcaatgcat gcgcgattgt
     4261 ggcgattgtt tgactattgt ttgtccattt catgacagac gccgcggcaa acaacgggag
     4321 cactcagctc cccgtcccgc actagtgcgg ccgcgaattc tctagagctt tttttatgat
     4381 tttactgaat tttttcttca catatcaccg aaatatgata tactattttt gtgaaatgct
     4441 gttaattagt atagcatgga tccgttttag agctgtgttg tttgttaaaa caacacagcg
     4501 agttaaaata aggcttagtc cgtactcaac ttgaaaaggt ggcaccgatt cggtgttttt
     4561 gtcgacgaat tcgcggccgc actagtccag gcatcaaata aaacgaaagg ctcagtcgaa
     4621 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     4681 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     4741 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
     4801 gctagcggca ctggccgtcg ttttacaacg ggcgcgcccg aattaattcc ttaaggaacg
     4861 tacagacggc ttaaaagcct ttaaaaacgt ttttaagggg tttgtagaca aggtaaagga
     4921 taaaacagca caattccaag aaaaacacga tttagaacct aaaaagaacg aatttgaact
     4981 aactcataac cgagaggtaa aaaaagaacg aagtcgagat cagggaatga gtttataaaa
     5041 taaaaaaagc acctgaaaag gtgtcttttt ttgatggttt tgaacttgtt ctttcttatc
     5101 ttgatacata tagaaataac gtcattttta ttttagttgc tgaaaggtgc gttgaagtgt
     5161 tggtatgtat gtgttttaaa gtattgaaaa cccttaaaat tggttgcaca gaaaaacccc
     5221 atctgttaaa gttataagtg actaaacaaa taactaaata gatgggggtt tcttttaata
     5281 ttatgtgtcc taatagtagc atttattcag atgaaaaatc aagggtttta gtggacaaga
     5341 caaaaagtgg aaaagtgaga ccatggagag aaaagaaaat cgctaatgtt gattactttg
     5401 aacttctgca tattcttgaa tttaaaaagg ctgaaagagt aaaagattgt gctgaaatat
     5461 tagagtataa acaaaatcgt gaaacaggcg aaagaaagtt gtatcgagtg tggttttgta
     5521 aatccaggct ttgtccaatg tgcaactgga ggagagcaat gaaacatggc attcagtcac
     5581 aaaaggttgt tgctgaagtt attaaacaaa agccaacagt tcgttggttg tttctcacat
     5641 taacagttaa aaatgtttat gatggcgaag aattaaataa gagtttgtca gatatggctc
     5701 aaggatttcg ccgaatgatg caatataaaa aaattaataa aaatcttgtt ggttttatgc
     5761 gtgcaacgga agtgacaata aataataaag ataattctta taatcagcac atgcatgtat
     5821 tggtatgtgt ggaaccaact tattttaaga atacagaaaa ctacgtgaat caaaaacaat
     5881 ggattcaatt ttggaaaaag gcaatgaaat tagactatga tccaaatgta aaagttcaaa
     5941 tgattcgacc gaaaaataaa tataaatcgg atatacaatc ggcaattgac gaaactgcaa
     6001 aatatcctgt aaaggatacg gattttatga ccgatgatga agaaaagaat ttgaaacgtt
     6061 tgtctgattt ggaggaaggt ttacaccgta aaaggttaat ctcctatggt ggtttgttaa
     6121 aagaaataca taaaaaatta aaccttgatg acacagaaga aggcgatttg attcatacag
     6181 atgatgacga aaaagccgat gaagatggat tttctattat tgcaatgtgg aattgggaac
     6241 ggaaaaatta ttttattaaa gagtagggcc ggcccagttt gttgaagatt agatgctata
     6301 attgttatta aaaggattga aggatgctta ggaagacgag ttattaatag ctgaataaga
     6361 acggtgctct ccaaatattc ttatttagaa aagcaaatct aaaattatct gaaaagggaa
     6421 tgagaatagt gaatggacca ataataatga ctagagaaga aagaatgaag attgttcatg
     6481 aaattaagga acgaatattg gataaatatg gggatgatgt taaggctatt ggtgtttatg
     6541 gctctcttgg tcgtcagact gatgggccct attcggatat tgagatgatg tgtgtcatgt
     6601 caacagagga agcagagttc agccatgaat ggacaaccgg tgagtggaag gtggaagtga
     6661 atttttatag cgaagagatt ctactagatt atgcatctca ggtggaatca gattggccgc
     6721 ttacacatgg tcaatttttc tctattttgc cgatttatga ttcaggtgga tacttagaga
     6781 aagtgtatca aactgctaaa tcggtagaag cccaaaagtt ccacgatgcg atttgtgccc
     6841 ttatcgtaga agagctgttt gaatatgcag gcaaatggcg taatattcgt gtgcaaggac
     6901 cgacaacatt tctaccatcc ttgactgtac aggtagcaat ggcaggtgcc atgttgattg
     6961 gtctgcatca tcgcatctgt tatacgacga gcgcttcggt cttaactgaa gcagttaagc
     7021 aatcagatct tccttcaggt tatgaccatc tgtgccagtt cgtaatgtct ggtcaacttt
     7081 ccgactctga gaaacttctg gaatcgctag agaatttctg gaatgggatt caggagtgga
     7141 cagaacgaca cggatatata gtggatgtgt caaaacgcat accattttga gtttaaactc
     7201 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     7261 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     7321 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     7381 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     7441 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     7501 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7561 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7621 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7681 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7741 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7801 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     7861 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     7921 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga aggcgagagt agggaactgc
     7981 caggcatcaa actaagc
//
