LOCUS       sequence-358235-        6109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  p11-lacY-wtx1 derivative toxic plasmid encoding an SpCas9 target 
            site for EGFP site 1 with an NGG PAM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6109)
  AUTHORS   Christie KA, Guo JA, Silverstein RA, Doll RM, Mabuchi M, Stutzman 
            HE, Lin J, Ma L, Walton RT, Pinello L, Robb GB, Kleinstiver BP
  TITLE     Precise DNA cleavage using CRISPR-SpRYgests.
  JOURNAL   Nat Biotechnol. 2022 Oct 6. pii: 10.1038/s41587-022-01492-y. doi: 
            10.1038/s41587-022-01492-y.
  PUBMED    36203014
REFERENCE   2  (bases 1 to 6109)
  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: "Nat 
            Biotechnol. 2022 Oct 6. pii: 10.1038/s41587-022-01492-y. doi: 
            10.1038/s41587-022-01492-y."
COMMENT     Sequence Label: pTox-EGFPsite1_NGG-PAM (BPK740)
FEATURES             Location/Qualifiers
     source          1..6109
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(161..1039)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1066..1350
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1273..1292
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1382..1687
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1566..1585
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1799..1816)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1969..2055
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2147..2174
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2193..2284
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2285..2353,2354..3145)
                     /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(2503..2522)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3187..3642
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3274..3293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3484..3505
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      3753..4341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4242..4261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4495..4512
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4677..4698
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4713..4718,4719..4736,4737..4743)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4751..4767
                     /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)."
     primer_bind     4756..4778
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4775..4791
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4775..4791
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4807..4823)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cacgcttagc ggcccgggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta
       61 aaggcctcga tgcataatgt gcctgtcaaa tggacgaagc agggattctg caaaccctat
      121 gctactccgt caagccgtca attgtctgat tcgttaccaa ttatgacaac ttgacggcta
      181 catcattcac tttttcttca caaccggcac ggaactcgct cgggctggcc ccggtgcatt
      241 ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga ccgacggtgg
      301 cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt tggtcctcgc
      361 gccagcttaa gacgctaatc cctaactgct ggcggaaaag atgtgacaga cgcgacggcg
      421 acaagcaaac atgctgtgcg acgctggcga tatcaaaatt gctgtctgcc aggtgatcgc
      481 tgatgtactg acaagcctcg cgtacccgat tatccatcgg tggatggagc gactcgttaa
      541 tcgcttccat gcgccgcagt aacaattgct caagcagatt tatcgccagc agctccgaat
      601 agcgcccttc cccttgcccg gcgttaatga tttgcccaaa caggtcgctg aaatgcggct
      661 ggtgcgcttc atccgggcga aagaaccccg tattggcaaa tattgacggc cagttaagcc
      721 attcatgcca gtaggcgcgc ggacgaaagt aaacccactg gtgataccat tcgcgagcct
      781 ccggatgacg accgtagtga tgaatctctc ctggcgggaa cagcaaaata tcacccggtc
      841 ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg accgcgaatg gtgagattga
      901 gaatataacc tttcattccc agcggtcggt cgataaaaaa atcgagataa ccgttggcct
      961 caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat
     1021 cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc
     1081 catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac
     1141 cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa
     1201 cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg
     1261 tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct
     1321 ttttatcgca actctctact gtttctccat acccgttttt ttgggctagc gattgaaaac
     1381 gatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     1441 acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg
     1501 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     1561 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     1621 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttttgggg
     1681 aatataatct agcattacgc tagggataac agggtaatat cacgctctag agggcacggg
     1741 cagcttgccg gtgggcatgc aagcttggct gttttggcgg atgagagaag attttcagcc
     1801 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca
     1861 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg
     1921 atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga
     1981 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     2041 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     2101 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg
     2161 acggatggcc tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa
     2221 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2281 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2341 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2401 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2461 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2521 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2581 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2641 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2701 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2761 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2821 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2881 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2941 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3001 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3061 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3121 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3181 tgatttacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3241 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3301 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3361 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     3421 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3481 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg
     3541 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3601 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttaaaaggat ctaggtgaag
     3661 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3721 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3781 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3841 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3901 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3961 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4021 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4081 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4141 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4201 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4261 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4321 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4381 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4441 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4501 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4561 ggtatttcac accgcatacg tacgatttaa ataggcctga ctcactatag ggagaccgga
     4621 attccctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     4681 gtgagttagc tcactcatta gggaccccgg gctttacact ttatgcttcc ggctcgtatg
     4741 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     4801 ggattcactg gccgtcgttt tacaacgtcg tgactggtaa aacccgggcg ttacccaact
     4861 taatcgcctt gcagcacatc cccctttcgc cagcaggcgt aataaggaaa ggatccatgt
     4921 actatttgaa aaacacaaac ttttggatgt tcggtttatt ctttttcttt tactttttta
     4981 tcatgggagc ctacttcccg tttttcccga tttggctaca tgatatcaac catatcagca
     5041 aaagtgatac gggtattatt tttgccgcta tttctctgtt ctcgctatta ttccaaccgc
     5101 tgtttggtct gctttctgac aaactcggtc tacgcaaata cctgctgtgg attattaccg
     5161 gcatgttagt gatgtttgcg ccgttcttta tttttatctt cgggccactg ctgcagtaca
     5221 acattttagt aggatcgatt gttggtggta tttatctagg ctttagtttt aacgccggtg
     5281 cgccagcagt agaggcattt attgagaaag tcagccggcg cagtaatttc gaatttggtc
     5341 gcgcgcggat gtttggcagt gttggctggg cgctggttgc ctcgattgtc gggatcatgt
     5401 tcaccattaa taatcagttt gttttctggc tgggctctgg cagttgtctc atcctcgccg
     5461 ttttactctt tttcgccaaa acggacgcgc cctcgagtgc cacggttgcc aatgcggtag
     5521 gtgccaacca ttcggcattt agccttaagc tggcactgga actgttcaga cagccaaaac
     5581 tgtggttttt gtcactgtat gttattggcg tttcctccac ctacgatgtt tttgaccaac
     5641 agtttgctaa tttctttact tcgttctttg ctaccggtga acagggtacc cgcgtatttg
     5701 gctacgtaac gacaatgggc gaattactta acgcctcgat tatgttcttt gcgccactga
     5761 tcattaatcg catcggtggg aagaatgccc tgctgctggc tggcactatt atgtctgtac
     5821 gtattattgg ctcatcgttc gccacctcag cgctggaagt ggttattctg aaaacgctgc
     5881 atatgtttga agtaccgttc ctgctggtgg gctcctttaa atatattact agtcagtttg
     5941 aagtgcgttt ttcagcgacg atttatctgg tcagtttcag cttctttaag caactggcga
     6001 tgatttttat gtctgtactg gcgggcaata tgtatgaaag cataggtttc caaggcgctt
     6061 atctggtgct gggtctggtg gcgctgggct tcaccttaat ttccgtgtt
//
