LOCUS       sequence-332572-        5235 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains the Cas12k gRNA for mutant library construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5235)
  AUTHORS   Chen W, Ren ZH, Tang N, Chai G, Zhang H, Zhang Y, Ma J, Wu Z, Shen 
            X, Huang X, Luo GZ, Ji Q
  TITLE     Targeted genetic screening in bacteria with a Cas12k-guided 
            transposase.
  JOURNAL   Cell Rep. 2021 Aug 31;36(9):109635. doi: 
            10.1016/j.celrep.2021.109635.
  PUBMED    34469724
REFERENCE   2  (bases 1 to 5235)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep.";
            date: "2021-08-31"; volume: "36(9):109635. doi"; pages: " 
            10.1016/j.celrep.2021.109635"
COMMENT     Sequence Label: pGTPA-lb
FEATURES             Location/Qualifiers
     source          1..5235
                     /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(361..379)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(362..379)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(397..413)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(397..413)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(410..432)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    421..437
                     /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(445..451,452..469,470..475))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    490..511
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(582..600)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        668..772
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(773..841,842..1633)
                     /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(991..1010)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1971..2322
                     /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      3626..4214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4115..4134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4368..4385
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4481..4936)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4618..4639)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4830..4849
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5062..5084
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5076..5093
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5077..5093
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5100..5119
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5100..5118
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5211..5220
                     /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 agacgattcg tctcattttt ttagccgtga
      301 tagtttgcga cagatatcga attcctgcag cccgggggat ccactagtaa gcttgagtat
      361 tctatagtgt cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
      421 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
      481 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
      541 gtcgggaaac ctgtcgtgcc agaattcttg aagacgaaag ggcctcgtga tacgcctatt
      601 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
      661 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      721 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
      781 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
      841 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
      901 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
      961 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
     1021 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1081 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1141 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1201 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1261 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     1321 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1381 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1441 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggat ctcgcggtat
     1501 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1561 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1621 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1681 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1741 cccttaacgt gagttttcgt tccactgagc gtcagacccc aattacacgc cactggctgt
     1801 gcttgctggg gtgacggtgg caacggtggc ggccttgctg ggctatcgcg ttggaaagaa
     1861 acgagggaaa ggggactgat aaaccggtct tagcccctcc ccttggtgtc caaccgctct
     1921 gtaggcctct caggcgccgc tggtgccgct ggttggacgc caagggtgaa tccgcctcga
     1981 taccctgatt actcgcttcc tgcgccctct caggcggcga taggggactg gtaaaacggg
     2041 gattgcccag acgcctcccc cgccccttca ggggcacaaa tgcggcccca acggggccac
     2101 gtagtggtgc gttttttgcg tttccaccct tttcttcctt ttccctttta aaccttttag
     2161 gacgtctaca ggccacgtaa tccgtggcct gtagagttta aaaagggacg gatttgttgc
     2221 cattaaggga cggatttgtt gttaagaagg gacggatttg ttgttgtaaa gggacggatt
     2281 tgttgtattg tgggacgcag atacagtgtc cccttataca caaggaatgt cgaacgtggc
     2341 ctcaccccca atggtttaca aaagcaatgc cctggtcgag gccgcgtatc gcctcagtgt
     2401 tcaggaacag cggatcgttc tggcctgtat tagccaggtg aagaggagcg agcctgtcac
     2461 cgatgaagtg atgtattcag tgacggcgga ggacatagcg acgatggcgg gtgtccctat
     2521 cgaatcttcc tacaaccagc tcaaagaagc ggccctgcgc ctgaaacggc gggaagtccg
     2581 gttaacccaa gagcccaatg gcaaggggaa aagaccgagt gtgatgatta ccggctgggt
     2641 gcaaacaatc atctaccggg agggtgaggg ccgtgtagaa ctcaggttca ccaaagacat
     2701 gctgccgtac ctgacggaac tcaccaaaca gttcaccaaa tacgccttgg ctgacgtggc
     2761 caagatggac agcacccacg cgatcaggct ttacgagctg ctcatgcaat gggacagcat
     2821 cggccagcgc gaaatagaaa ttgaccagct gcgaaagtgg tttcaactgg aaggccggta
     2881 tccctcgatc aaggacttca agttgcgagt gcttgatcca gccgtgacgc agatcaacga
     2941 gcacagcccg ctacaggtgg agtgggcgca gcgaaagacc gggcgcaagg tcacacatct
     3001 gttgttcagt tttggaccga agaagcccgc caaggcggtg ggtaaggccc cagcgaagcg
     3061 caaggccggg aagatttcag atgctgagat cgcgaaacag gctcgccctg gtgagacatg
     3121 ggaagcggcc cgcgctcgac taacccagat gccgcttgcg caaactatta actggcgaac
     3181 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3241 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3301 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3361 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3421 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3481 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3541 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3601 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3661 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3721 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3781 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3841 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3901 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3961 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4021 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4081 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4141 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4201 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4261 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4321 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4381 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4441 cacaccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt
     4501 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4561 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     4621 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4681 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     4741 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4801 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4861 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4921 ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     4981 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     5041 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
     5101 aatacgactc actatagggc gaattcgagc tcggtacccg gggatccgtc gacgccttgc
     5161 tcaaattgga atcaggtttg tgccaatacc agtagaaaca gacgaagaat ccatggcggc
     5221 cgctgtcatg ggccc
//
