LOCUS       sequence-340981-        8417 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Plasmid contains the Cas12k gRNA and transposon donor cassette 
            assembled with gentamycin-resistant gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8417)
  TITLE     Genetic manipulation in P. aeruginosa and Mycobacterium tuberculosis
REFERENCE   2  (bases 1 to 8417)
  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-Gm
FEATURES             Location/Qualifiers
     source          1..8417
                     /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        join(634..639,640..656,657..662)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             851..1384
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(1690..1708)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1691..1708)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1726..1742)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1726..1742)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1739..1761)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1750..1766
                     /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(1774..1780,1781..1798,1799..1804))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1819..1840
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1911..1929)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1997..2101
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2102..2170,2171..2962)
                     /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(2320..2339)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3300..3651
                     /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      4955..5543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5444..5463
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5697..5714
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5810..6265)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5947..5968)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6159..6178
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6391..6413
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6405..6422
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6406..6422
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6429..6448
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6429..6447
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6477..6496
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(6535..7869,7870..7956))
                     /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      8393..8402
                     /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 ctcgaggaat aggaacttcg gaataggaac ttcaagatcc
      601 cctgattccc tttgtcaaca gcaatggatc gaattgacat aagcctgttc ggttcgtaaa
      661 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
      721 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tgtacagtct
      781 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg
      841 agcagcaacg atgttacgca gcagcaacga tgttacgcag cagggcagtc gccctaaaac
      901 aaagttaggt ggctcaagta tgggcatcat tcgcacatgt aggctcggcc ctgaccaagt
      961 caaatccatg cgggctgctc ttgatctttt cggtcgtgag ttcggagacg tagccaccta
     1021 ctcccaacat cagccggact ccgattacct cgggaacttg ctccgtagta agacattcat
     1081 cgcgcttgct gccttcgacc aagaagcggt tgttggcgct ctcgcggctt acgttctgcc
     1141 caagtttgag cagccgcgta gtgagatcta tatctatgat ctcgcagtct ccggcgagca
     1201 ccggaggcag ggcattgcca ccgcgctcat caatctcctc aagcatgagg ccaacgcgct
     1261 tggtgcttat gtgatctacg tgcaagcaga ttacggtgac gatcccgcag tggctctcta
     1321 tacaaagttg ggcatacggg aagaagtgat gcactttgat atcgacccaa gtaccgccac
     1381 ctaacaattc gttcaagccg agatcagctt cccggccgcg gagttgttcg gtaaattgtc
     1441 acaacgccgc ggccaattcg atctagatca gtaactatta aacttagggg tgggttgaaa
     1501 gcaagtcctt ttatccgctt gttttaattg ctttgtataa taattgcaga gcatattata
     1561 ttgatgacat ttaatttgtc atcaattaat taagcaacgc tgatgggtca cgacgacaat
     1621 taaatagtca caatgacatt aatctgtcac cgacgacaga taatttgtca ctgtacaaag
     1681 cttgagtatt ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc
     1741 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     1801 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     1861 cgctttccag tcgggaaacc tgtcgtgcca gaattcttga agacgaaagg gcctcgtgat
     1921 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     1981 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2041 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2101 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2161 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2221 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2281 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2341 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2401 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2461 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2521 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2581 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2641 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2701 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2761 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggatc
     2821 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2881 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2941 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3001 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3061 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagacccca attacacgcc
     3121 actggctgtg cttgctgggg tgacggtggc aacggtggcg gccttgctgg gctatcgcgt
     3181 tggaaagaaa cgagggaaag gggactgata aaccggtctt agcccctccc cttggtgtcc
     3241 aaccgctctg taggcctctc aggcgccgct ggtgccgctg gttggacgcc aagggtgaat
     3301 ccgcctcgat accctgatta ctcgcttcct gcgccctctc aggcggcgat aggggactgg
     3361 taaaacgggg attgcccaga cgcctccccc gccccttcag gggcacaaat gcggccccaa
     3421 cggggccacg tagtggtgcg ttttttgcgt ttccaccctt ttcttccttt tcccttttaa
     3481 accttttagg acgtctacag gccacgtaat ccgtggcctg tagagtttaa aaagggacgg
     3541 atttgttgcc attaagggac ggatttgttg ttaagaaggg acggatttgt tgttgtaaag
     3601 ggacggattt gttgtattgt gggacgcaga tacagtgtcc ccttatacac aaggaatgtc
     3661 gaacgtggcc tcacccccaa tggtttacaa aagcaatgcc ctggtcgagg ccgcgtatcg
     3721 cctcagtgtt caggaacagc ggatcgttct ggcctgtatt agccaggtga agaggagcga
     3781 gcctgtcacc gatgaagtga tgtattcagt gacggcggag gacatagcga cgatggcggg
     3841 tgtccctatc gaatcttcct acaaccagct caaagaagcg gccctgcgcc tgaaacggcg
     3901 ggaagtccgg ttaacccaag agcccaatgg caaggggaaa agaccgagtg tgatgattac
     3961 cggctgggtg caaacaatca tctaccggga gggtgagggc cgtgtagaac tcaggttcac
     4021 caaagacatg ctgccgtacc tgacggaact caccaaacag ttcaccaaat acgccttggc
     4081 tgacgtggcc aagatggaca gcacccacgc gatcaggctt tacgagctgc tcatgcaatg
     4141 ggacagcatc ggccagcgcg aaatagaaat tgaccagctg cgaaagtggt ttcaactgga
     4201 aggccggtat ccctcgatca aggacttcaa gttgcgagtg cttgatccag ccgtgacgca
     4261 gatcaacgag cacagcccgc tacaggtgga gtgggcgcag cgaaagaccg ggcgcaaggt
     4321 cacacatctg ttgttcagtt ttggaccgaa gaagcccgcc aaggcggtgg gtaaggcccc
     4381 agcgaagcgc aaggccggga agatttcaga tgctgagatc gcgaaacagg ctcgccctgg
     4441 tgagacatgg gaagcggccc gcgctcgact aacccagatg ccgcttgcgc aaactattaa
     4501 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4561 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4621 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4681 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4741 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4801 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4861 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4921 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4981 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5041 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5101 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5161 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5221 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5281 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5341 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5401 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5461 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5521 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5581 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5641 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5701 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     5761 gcggtatttc acaccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     5821 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     5881 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     5941 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     6001 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     6061 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     6121 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     6181 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     6241 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     6301 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     6361 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     6421 gtgaattgta atacgactca ctatagggcg aattcgagct cggtacccgg ggatcccggc
     6481 atcagagcag attgtactga gagtgcacca taatcggcat tttcttttgc gtttttattt
     6541 gttaactgtt aattgtcctt gttcaaggat gctgtctttg acaacagatg ttttcttgcc
     6601 tttgatgttc agcaggaagc taggcgcaaa cgttgattgt ttgtctgcgt agaatcctct
     6661 gtttgtcata tagcttgtaa tcacgacatt gtttcctttc gcttgaggta cagcgaagtg
     6721 tgagtaagta aaggttacat cgttaggatc aagatccatt tttaacacaa ggccagtttt
     6781 gttcagcggc ttgtatgggc cagttaaaga attagaaaca taaccaagca tgtaaatatc
     6841 gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga acagatacca
     6901 tttgccgttc attttaaaga cgttcgcgcg ttcaatttca tctgttactg tgttagatgc
     6961 aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa tcataccgag
     7021 agcgccgttt gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc
     7081 ttgacggaag aatgatgtgc ttttgccata gtatgctttg ttaaataaag attcttcgcc
     7141 ttggtagcca tcttcagttc cagtgtttgc ttcaaatact aagtatttgt ggcctttatc
     7201 ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc
     7261 gatgaactgc tgtacatttt gatacgtttt tccgtcaccg tcaaagattg atttataatc
     7321 ctctacaccg ttgatgttca aagagctgtc tgatgctgat acgttaactt gtgcagttgt
     7381 cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg tagaataaac ggatttttcc
     7441 gtcagatgta aatgtggctg aacctgacca ttcttgtgtt tggtctttta ggatagaatc
     7501 atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat
     7561 agaagtttcg ccgacttttt gatagaacat gtaaatcgat gtgtcatccg catttttagg
     7621 atctccggct aatgcaaaga cgatgtggta gccgtgatag tttgcgacag tgccgtcagc
     7681 gttttgtaat ggccagctgt cccaaacgtc caggcctttt gcagaagaga tatttttaat
     7741 tgtggacgaa tcgaactcag gaacttgata tttttcattt ttttgctgtt cagggatttg
     7801 cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg gcttttggtt
     7861 cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag taaaggttaa
     7921 tactgttgct tgttttgcaa actttttgat gttcatcgtt catgtctcct tttttatgta
     7981 ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa gatggcaagt tagttacgca
     8041 caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa agtatacact ttgcccttta
     8101 cacattttag gtcttgcctg ctttatcagt aacaaacccg cgcgatttac ttttcgacct
     8161 cattctatta gactctcgtt tggattgcaa ctggtctatt ttcctctttt gtttgataga
     8221 aaatcataaa aggatttgca gactacgggc ctaaagaact aaaaaatcta tctgtttctt
     8281 ttcattctct gtatttttta tagtttctgt tgcatgggca taaagttgcg tcgacgcctt
     8341 gctcaaattg gaatcaggtt tgtgccaata ccagtagaaa cagacgaaga atccatggcg
     8401 gccgctgtca tgggccc
//
