LOCUS       sequence-340942-        7570 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Plasmid contains the Cas12k gRNA and transposon donor cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7570)
  TITLE     Genetic manipulation in P. aeruginosa and Mycobacterium tuberculosis
REFERENCE   2  (bases 1 to 7570)
  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"
COMMENT     Sequence Label: pGTPA
FEATURES             Location/Qualifiers
     source          1..7570
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..35
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     promoter        complement(843..861)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(844..861)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(879..895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(879..895)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(892..914)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    903..919
                     /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)."
     promoter        complement(join(927..933,934..951,952..957))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    972..993
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1064..1082)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1150..1254
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1255..1323,1324..2115)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1473..1492)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2453..2804
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     rep_origin      4108..4696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4597..4616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4850..4867
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4963..5418)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5100..5121)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5312..5331
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5544..5566
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5558..5575
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5559..5575
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5582..5601
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5582..5600
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5630..5649
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(5688..7022,7023..7109))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     regulatory      7546..7555
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ttgacagcta gctcagtcct aggtataata ctagtatatt aatagcgccg caattcatgc
       61 tgcttgcagc ctctgaattt tgttaaatga gggttagttt gactgtataa atacagtctt
      121 gctttctgac cctggtagct gctcaccctg atgctgctgt caatagacag gataggtgcg
      181 ctcccagcaa taagggcgcg gatgtactgc tgtagtggct actgaatcac ccccgatcaa
      241 gggggaaccc tccaaaaggt gggttgaaag agaccattgg tctcattttt ttagccgtga
      301 tagtttgcga cagatatcga attcctgcag cccgggggat ccactagttg tacagtgact
      361 aattatatgt cgttgtgaca aattattgtc atcagtaaaa tccttataca gtatagatta
      421 tagcgctttg gcagttttag cataacctct ttgcagtgac aaaatagatg tcgttgtccg
      481 tgattgtgac aaattagctg tcgctttgca agataggaaa aagcttttgt gtattttcat
      541 aatgacaaat tgactgtcgc ctcgagccaa gctcggcgcg ccattgggat gagctccacc
      601 gcggtggcgg ccgctctaga tcagtaacta ttaaacttag gggtgggttg aaagcaagtc
      661 cttttatccg cttgttttaa ttgctttgta taataattgc agagcatatt atattgatga
      721 catttaattt gtcatcaatt aattaagcaa cgctgatggg tcacgacgac aattaaatag
      781 tcacaatgac attaatctgt caccgacgac agataatttg tcactgtaca aagcttgagt
      841 attctatagt gtcacctaaa tagcttggcg taatcatggt catagctgtt tcctgtgtga
      901 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
      961 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     1021 cagtcgggaa acctgtcgtg ccagaattct tgaagacgaa agggcctcgt gatacgccta
     1081 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     1141 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1201 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     1261 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1321 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1381 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1441 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     1501 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1561 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1621 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1681 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1741 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     1801 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1861 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1921 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg atctcgcggt
     1981 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2041 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2101 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     2161 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2221 atcccttaac gtgagttttc gttccactga gcgtcagacc ccaattacac gccactggct
     2281 gtgcttgctg gggtgacggt ggcaacggtg gcggccttgc tgggctatcg cgttggaaag
     2341 aaacgaggga aaggggactg ataaaccggt cttagcccct ccccttggtg tccaaccgct
     2401 ctgtaggcct ctcaggcgcc gctggtgccg ctggttggac gccaagggtg aatccgcctc
     2461 gataccctga ttactcgctt cctgcgccct ctcaggcggc gataggggac tggtaaaacg
     2521 gggattgccc agacgcctcc cccgcccctt caggggcaca aatgcggccc caacggggcc
     2581 acgtagtggt gcgttttttg cgtttccacc cttttcttcc ttttcccttt taaacctttt
     2641 aggacgtcta caggccacgt aatccgtggc ctgtagagtt taaaaaggga cggatttgtt
     2701 gccattaagg gacggatttg ttgttaagaa gggacggatt tgttgttgta aagggacgga
     2761 tttgttgtat tgtgggacgc agatacagtg tccccttata cacaaggaat gtcgaacgtg
     2821 gcctcacccc caatggttta caaaagcaat gccctggtcg aggccgcgta tcgcctcagt
     2881 gttcaggaac agcggatcgt tctggcctgt attagccagg tgaagaggag cgagcctgtc
     2941 accgatgaag tgatgtattc agtgacggcg gaggacatag cgacgatggc gggtgtccct
     3001 atcgaatctt cctacaacca gctcaaagaa gcggccctgc gcctgaaacg gcgggaagtc
     3061 cggttaaccc aagagcccaa tggcaagggg aaaagaccga gtgtgatgat taccggctgg
     3121 gtgcaaacaa tcatctaccg ggagggtgag ggccgtgtag aactcaggtt caccaaagac
     3181 atgctgccgt acctgacgga actcaccaaa cagttcacca aatacgcctt ggctgacgtg
     3241 gccaagatgg acagcaccca cgcgatcagg ctttacgagc tgctcatgca atgggacagc
     3301 atcggccagc gcgaaataga aattgaccag ctgcgaaagt ggtttcaact ggaaggccgg
     3361 tatccctcga tcaaggactt caagttgcga gtgcttgatc cagccgtgac gcagatcaac
     3421 gagcacagcc cgctacaggt ggagtgggcg cagcgaaaga ccgggcgcaa ggtcacacat
     3481 ctgttgttca gttttggacc gaagaagccc gccaaggcgg tgggtaaggc cccagcgaag
     3541 cgcaaggccg ggaagatttc agatgctgag atcgcgaaac aggctcgccc tggtgagaca
     3601 tgggaagcgg cccgcgctcg actaacccag atgccgcttg cgcaaactat taactggcga
     3661 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3721 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3781 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3841 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3901 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3961 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4021 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4081 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4141 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4201 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     4261 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4321 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4381 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4441 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4501 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4561 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4621 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4681 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4741 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4801 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4861 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4921 ttcacaccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata
     4981 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5041 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5101 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5161 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5221 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5281 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5341 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5401 cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     5461 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     5521 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
     5581 gtaatacgac tcactatagg gcgaattcga gctcggtacc cggggatccc ggcatcagag
     5641 cagattgtac tgagagtgca ccataatcgg cattttcttt tgcgttttta tttgttaact
     5701 gttaattgtc cttgttcaag gatgctgtct ttgacaacag atgttttctt gcctttgatg
     5761 ttcagcagga agctaggcgc aaacgttgat tgtttgtctg cgtagaatcc tctgtttgtc
     5821 atatagcttg taatcacgac attgtttcct ttcgcttgag gtacagcgaa gtgtgagtaa
     5881 gtaaaggtta catcgttagg atcaagatcc atttttaaca caaggccagt tttgttcagc
     5941 ggcttgtatg ggccagttaa agaattagaa acataaccaa gcatgtaaat atcgttagac
     6001 gtaatgccgt caatcgtcat ttttgatccg cgggagtcag tgaacagata ccatttgccg
     6061 ttcattttaa agacgttcgc gcgttcaatt tcatctgtta ctgtgttaga tgcaatcagc
     6121 ggtttcatca cttttttcag tgtgtaatca tcgtttagct caatcatacc gagagcgccg
     6181 tttgctaact cagccgtgcg ttttttatcg ctttgcagaa gtttttgact ttcttgacgg
     6241 aagaatgatg tgcttttgcc atagtatgct ttgttaaata aagattcttc gccttggtag
     6301 ccatcttcag ttccagtgtt tgcttcaaat actaagtatt tgtggccttt atcttctacg
     6361 tagtgaggat ctctcagcgt atggttgtcg cctgagctgt agttgccttc atcgatgaac
     6421 tgctgtacat tttgatacgt ttttccgtca ccgtcaaaga ttgatttata atcctctaca
     6481 ccgttgatgt tcaaagagct gtctgatgct gatacgttaa cttgtgcagt tgtcagtgtt
     6541 tgtttgccgt aatgtttacc ggagaaatca gtgtagaata aacggatttt tccgtcagat
     6601 gtaaatgtgg ctgaacctga ccattcttgt gtttggtctt ttaggataga atcatttgca
     6661 tcgaatttgt cgctgtcttt aaagacgcgg ccagcgtttt tccagctgtc aatagaagtt
     6721 tcgccgactt tttgatagaa catgtaaatc gatgtgtcat ccgcattttt aggatctccg
     6781 gctaatgcaa agacgatgtg gtagccgtga tagtttgcga cagtgccgtc agcgttttgt
     6841 aatggccagc tgtcccaaac gtccaggcct tttgcagaag agatattttt aattgtggac
     6901 gaatcgaact caggaacttg atatttttca tttttttgct gttcagggat ttgcagcata
     6961 tcatggcgtg taatatggga aatgccgtat gtttccttat atggcttttg gttcgtttct
     7021 ttcgcaaacg cttgagttgc gcctcctgcc agcagtgcgg tagtaaaggt taatactgtt
     7081 gcttgttttg caaacttttt gatgttcatc gttcatgtct ccttttttat gtactgtgtt
     7141 agcggtctgc ttcttccagc cctcctgttt gaagatggca agttagttac gcacaataaa
     7201 aaaagaccta aaatatgtaa ggggtgacgc caaagtatac actttgccct ttacacattt
     7261 taggtcttgc ctgctttatc agtaacaaac ccgcgcgatt tacttttcga cctcattcta
     7321 ttagactctc gtttggattg caactggtct attttcctct tttgtttgat agaaaatcat
     7381 aaaaggattt gcagactacg ggcctaaaga actaaaaaat ctatctgttt cttttcattc
     7441 tctgtatttt ttatagtttc tgttgcatgg gcataaagtt gcgtcgacgc cttgctcaaa
     7501 ttggaatcag gtttgtgcca ataccagtag aaacagacga agaatccatg gcggccgctg
     7561 tcatgggccc
//
