LOCUS       62056_17560        7196 bp DNA     circular SYN 28-JUL-2021
DEFINITION  Cloning vector pMTL575555, complete sequence.
ACCESSION   MZ182078
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7196)
  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 7196)
  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 7196)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol"; date: "2021"; volume: "10"; issue: "7"; pages: "1739-1749"
COMMENT     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"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7196
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(12..3374)
                     /label=St1Cas9
                     /note="Cas9 endonuclease from the Streptococcus
                     thermophilus Type II CRISPR/Cas system (Esvelt et al., 
                     2013)"
     regulatory      complement(3375..3559)
                     /label=lactate dehydrogenase promoter and RBS
                     /note="lactate dehydrogenase promoter and RBS"
                     /regulatory_class="promoter"
     regulatory      3566..3656
                     /label=glyceraldehyde 3-phosphate dehydrogenase promoter
                     /note="glyceraldehyde 3-phosphate dehydrogenase promoter"
                     /regulatory_class="promoter"
     terminator      3794..3880
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3972..3999
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     primer_bind     complement(4009..4025)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4461..5462
                     /label=repB
                     /note="RepB replication protein"
     CDS             5619..6389
                     /codon_start=1
                     /transl_table=11
                     /product="kanamycin nucleotidyltransferase"
                     /label=kanamycin nucleotidyltransferase
                     /protein_id="QXV87316.1"
                     /translation="MRIVNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGR
                     QTDGPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFYSEEILLDYASQVESDWPLTHG
                     QFFSILPIYDSGGYLEKVYQTAKSVEAQKFHDAICALIVEELFEYAGKWRNIRVQGPTT
                     FLPSLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSD
                     SEKLLESLENFWNGIQEWTERHGYIVDVSKRIPF"
     rep_origin      6397..6980
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggtt aaaaatctag cttaggctta tcaccctcat ttttgatgat atgctgattt
       61 cctaggacat ctgttcttac cttataaata gaaatatttg attttcctag cccttttttg
      121 cattgaccac tattagcaac gttacccaac actttaatta acgcctcacc tccttcaaat
      181 ttctgtttat cataaggttt taattcaaca taatgctttt gtttaggcat agttcgagaa
      241 agaaaacgga aaagctgttg ttcttttgtt tctgtatctt taacgagtaa caaatcattt
      301 ttataaagtg taaacttgaa ttctgaatca gaatctacac cctctttttt cttaatgtca
      361 ttgtattttt cctgggaaat cttatatgtt cctgtccctt tttcaaattg taaatcagca
      421 tatttcaatc cgaggatttc gtattttcca gtagtcttat tgaaatagac atccgctctc
      481 catggagaaa ctgactgtaa gacaacttta ttgttactat ccttcggagt aatatcaata
      541 tgattgccta gcttactatc atagtattta agactcttga tttcaggacc attgcctttt
      601 ttactatatt tacgaatata gccatgttct tctttatatt ttaggaaagg attacatggt
      661 acctctttcc ctttttcatt tatttgctta ttaggatagt tctctaaaat tggctcgata
      721 actttctcaa aggtttgtgg gtcgtgacga tacatgagga attttgactt atccttctta
      781 taaatcttca taaaggcatc ataaccatcc tgagtataga tatctttgat tttccctaag
      841 acataagttt catccgcctt atcttttcct actttagcct gtcttgtcgc ataaatagtg
      901 gcatctgata ttttacgatt aaacttagaa tccacttgat aagagaataa gatactgtct
      961 tcaaattctt tactcttcaa tgtatcaaca aaatgttgat aaggggcttt gaacacagat
     1021 tccttgtact catcatcact aataagttca cctgtttcaa tatcaaggag ttggtcttct
     1081 gaataactta caagggtatt cttttgtttt ttccacaaat tcaactgact tgaggcggca
     1141 ataatcaatg catcgacagc atggtgatga taagtatcac gagtcttctc aattccccaa
     1201 tggcgtctca attgagatgt aaattggcca cgaaccacgg aaacttttgt atcaatcttg
     1261 tgagctctaa agtgttcttg aagggcattg aggacaactc ttgaagcgta tcttgtatct
     1321 acaagatttc gttcaataaa tttctttcga acatcaaact ttgaaatatc ttcttctgta
     1381 aggaggtatt cttttttctt gttcgaaagt gtttttgact cacgtacaaa agcttttaat
     1441 tcacggaaag accacgcatc atccatacta tctaaagcct gataaggtgt tcgttgtcct
     1501 ttttcttggt tagcagttgc ataaaccaaa accttatttg caaggctatc atcgaatgtg
     1561 atagaaagag gtaaaatatg atctacttca aactgattag aattatttat caaatcatgg
     1621 attgagattg tcttaccagt ataaaggcaa cgttctcctt gctgatgcca aaggcggatt
     1681 ttagtcgcta attgcttatg accgtggaaa acactatgtg gtaattcagc ctttccatta
     1741 tattggttag cagccttaag cattgctgca tctttttcat ctttgttggc tttttgaatc
     1801 ttttgaatag ctttcttttc atcatcttca tttgtttcac gagccatttc gatgacaata
     1861 ttgtcaaagt ctccgtattc tttaatcgcc gcatttacga tttttatagc ctggcgaaca
     1921 gacttagcaa caacaggatt atagatttct tcagttaata gtttttcatc tatatatttt
     1981 gttttatttg aagacgaagt cgttttttgt tttccaagtc gtgttaggat agtcatttgc
     2041 tcttctgacg tctcatacaa ttctggaatt aactccatca tcagtttgac agaaaaatta
     2101 tgccatcctt ttccaaaaat ggaactattt gctttgcgga attgaaccaa ttcgtcaact
     2161 tgcttctggc taaagctacc atcagcaaat tcatgttcta aggcttcttg aataccttcc
     2221 ctctcagtgt ttaatgttaa gacataggct aatttatcaa gcgtttctct atccatttgc
     2281 tcaatatcta aggtttcaag cgttttcatt tttcgatagg cttcgaaagt atgaatctca
     2341 gccttacctg atttgtcgat acggtatccc ttgatatctg caacatcaca agaaagtaac
     2401 ttagcgatat atttaaaaag tttcgctggc cccattgcct tttcattttt gacataatta
     2461 atgatttgat tcttctgttc tttgctcaac tttttggttt cagtaggaac tgttagattg
     2521 ttcaaatcat ttagcaaatt gaattcttga gccgtgtagg aagcttttgc tgctctaaac
     2581 tcgtctggat aaaatgtaca tttcccaatt agaattccaa aaatattgtc taaagtttct
     2641 ccactcgttc tgtaacgacc ataatcagtc cgtgactttt catttccggg tccatgataa
     2701 tatttccgtt ttccagttaa aatttcgaga taacgattaa taaattcatc tgtaatctgt
     2761 ggattaaatt cttgttgagt ttgcagtatc cttaaggctt ctgaacgata agctgatgtt
     2821 ggaaagacat taatcaagcg atgttttttg ccatctttct caacagtaaa atcaccacgt
     2881 aattgaccat atgtttggta gcgttccaac tgtatctgtc ccggtgtctt agtttctaat
     2941 tgtttactat tttccttaac aatttgtgca tagtctccta ctgatgaatt tccgtcatca
     3001 ctagcatcat cgaggtaact aatcccacgg tgtttcacca tatttttaag agcgataaac
     3061 agttcttcat tagacaattc atcggtcaag cccttaactc gtaattgata tgggttaaga
     3121 ttaattgaaa tcttcgtaaa atcagtgatt aatccacttt cctcaaatag acgatttaaa
     3181 cgaactctac gatgtttttt acgtcgtgcc aagcgtcttc cttgacgatt cgttctacgt
     3241 actaggttat tttccgcttg agctgctggg aagatgcgtg agtttttatg gataatttct
     3301 cctgtcactt tgttaaggat acccacacca acagaaccta taccgatatc aagtcctaaa
     3361 actaagtcac tcattgcatt catcctccct caatataatg cgaacacttt catataaata
     3421 ttttacgcga aaggcgcacc gcaatgcatg cgcgattgtg gcgattgttt gactattgtt
     3481 tgtccatttc atgacagacg ccgcggcaaa caacgggagc actcagctcc ccgtcccgca
     3541 ctagtgcggc cgcgaattct ctagagcttt ttttatgatt ttactgaatt ttttcttcac
     3601 atatcaccga aatatgatat actatttttg tgaaatgctg ttaattagta tagcatggat
     3661 ccgtttttgt actctcaaga ttcaataatc ttgcagaagc tacaaagata aggcttcatg
     3721 ccgaaatcaa caccctgtca ttttatggca gggtgttttg tcgacgaatt cgcggccgca
     3781 ctagtccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     3841 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
     3901 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca
     3961 tcaaattaag cagaaggcca tcctgacgga tggccttttg ctagcggcac tggccgtcgt
     4021 tttacaacgg gcgcgcccga attaattcct taaggaacgt acagacggct taaaagcctt
     4081 taaaaacgtt tttaaggggt ttgtagacaa ggtaaaggat aaaacagcac aattccaaga
     4141 aaaacacgat ttagaaccta aaaagaacga atttgaacta actcataacc gagaggtaaa
     4201 aaaagaacga agtcgagatc agggaatgag tttataaaat aaaaaaagca cctgaaaagg
     4261 tgtctttttt tgatggtttt gaacttgttc tttcttatct tgatacatat agaaataacg
     4321 tcatttttat tttagttgct gaaaggtgcg ttgaagtgtt ggtatgtatg tgttttaaag
     4381 tattgaaaac ccttaaaatt ggttgcacag aaaaacccca tctgttaaag ttataagtga
     4441 ctaaacaaat aactaaatag atgggggttt cttttaatat tatgtgtcct aatagtagca
     4501 tttattcaga tgaaaaatca agggttttag tggacaagac aaaaagtgga aaagtgagac
     4561 catggagaga aaagaaaatc gctaatgttg attactttga acttctgcat attcttgaat
     4621 ttaaaaaggc tgaaagagta aaagattgtg ctgaaatatt agagtataaa caaaatcgtg
     4681 aaacaggcga aagaaagttg tatcgagtgt ggttttgtaa atccaggctt tgtccaatgt
     4741 gcaactggag gagagcaatg aaacatggca ttcagtcaca aaaggttgtt gctgaagtta
     4801 ttaaacaaaa gccaacagtt cgttggttgt ttctcacatt aacagttaaa aatgtttatg
     4861 atggcgaaga attaaataag agtttgtcag atatggctca aggatttcgc cgaatgatgc
     4921 aatataaaaa aattaataaa aatcttgttg gttttatgcg tgcaacggaa gtgacaataa
     4981 ataataaaga taattcttat aatcagcaca tgcatgtatt ggtatgtgtg gaaccaactt
     5041 attttaagaa tacagaaaac tacgtgaatc aaaaacaatg gattcaattt tggaaaaagg
     5101 caatgaaatt agactatgat ccaaatgtaa aagttcaaat gattcgaccg aaaaataaat
     5161 ataaatcgga tatacaatcg gcaattgacg aaactgcaaa atatcctgta aaggatacgg
     5221 attttatgac cgatgatgaa gaaaagaatt tgaaacgttt gtctgatttg gaggaaggtt
     5281 tacaccgtaa aaggttaatc tcctatggtg gtttgttaaa agaaatacat aaaaaattaa
     5341 accttgatga cacagaagaa ggcgatttga ttcatacaga tgatgacgaa aaagccgatg
     5401 aagatggatt ttctattatt gcaatgtgga attgggaacg gaaaaattat tttattaaag
     5461 agtagggccg gcccagtttg ttgaagatta gatgctataa ttgttattaa aaggattgaa
     5521 ggatgcttag gaagacgagt tattaatagc tgaataagaa cggtgctctc caaatattct
     5581 tatttagaaa agcaaatcta aaattatctg aaaagggaat gagaatagtg aatggaccaa
     5641 taataatgac tagagaagaa agaatgaaga ttgttcatga aattaaggaa cgaatattgg
     5701 ataaatatgg ggatgatgtt aaggctattg gtgtttatgg ctctcttggt cgtcagactg
     5761 atgggcccta ttcggatatt gagatgatgt gtgtcatgtc aacagaggaa gcagagttca
     5821 gccatgaatg gacaaccggt gagtggaagg tggaagtgaa tttttatagc gaagagattc
     5881 tactagatta tgcatctcag gtggaatcag attggccgct tacacatggt caatttttct
     5941 ctattttgcc gatttatgat tcaggtggat acttagagaa agtgtatcaa actgctaaat
     6001 cggtagaagc ccaaaagttc cacgatgcga tttgtgccct tatcgtagaa gagctgtttg
     6061 aatatgcagg caaatggcgt aatattcgtg tgcaaggacc gacaacattt ctaccatcct
     6121 tgactgtaca ggtagcaatg gcaggtgcca tgttgattgg tctgcatcat cgcatctgtt
     6181 atacgacgag cgcttcggtc ttaactgaag cagttaagca atcagatctt ccttcaggtt
     6241 atgaccatct gtgccagttc gtaatgtctg gtcaactttc cgactctgag aaacttctgg
     6301 aatcgctaga gaatttctgg aatgggattc aggagtggac agaacgacac ggatatatag
     6361 tggatgtgtc aaaacgcata ccattttgag tttaaactcc tttttttctg cgcgtaatct
     6421 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6481 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6541 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6601 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6661 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6721 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6781 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6841 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6901 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6961 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7021 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     7081 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     7141 cagtgagcga ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa ctaagc
//
