LOCUS       sequence-358237-        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 NGA 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_NGA-PAM (BPK754)
FEATURES             Location/Qualifiers
     source          1..6109
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(124..1002)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1029..1313
                     /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     1236..1255
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1345..1650
                     /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     1529..1548
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1762..1779)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1932..2018
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2110..2137
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2156..2247
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2248..2316,2317..3108)
                     /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(2466..2485)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3150..3605
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3237..3256)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3447..3468
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      3716..4304
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4205..4224
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4458..4475
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4640..4661
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4676..4681,4682..4699,4700..4706)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4714..4730
                     /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     4719..4741
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4738..4754
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4738..4754
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4770..4786)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cgtcaggtga atgaagtcgc ttaaaggcct cgatgcataa tgtgcctgtc aaatggacga
       61 agcagggatt ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct gattcgttac
      121 caattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc
      181 gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa
      241 accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc
      301 ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa
      361 aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa
      421 attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat
      481 cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag
      541 atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc
      601 aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc
      661 aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca
      721 ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg
      781 gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc
      841 ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa
      901 aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa
      961 cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc
     1021 attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta
     1081 ctggctcttc tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag
     1141 cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt
     1201 ccacattgat tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag
     1261 attagcggat cctacctgac gctttttatc gcaactctct actgtttctc catacccgtt
     1321 tttttgggct agcgattgaa aacgatgcag tttaaggttt acacctataa aagagagagc
     1381 cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg
     1441 gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg
     1501 gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg
     1561 gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac
     1621 gccattaacc tgatgttttg gggaatataa tctagcatta cgctagggat aacagggtaa
     1681 tatcacgctc tagagggcac gggcagcttg ccggtgagca tgcaagcttg gctgttttgg
     1741 cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat
     1801 aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     1861 agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa
     1921 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     1981 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     2041 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     2101 aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttttgt
     2161 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2221 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     2281 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2341 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2401 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2461 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     2521 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2581 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2641 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2701 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2761 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     2821 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2881 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2941 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     3001 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3061 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3121 gtttactcat atatacttta gattgattta cgcgccctgt agcggcgcat taagcgcggc
     3181 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3241 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3301 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3361 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3421 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3481 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3541 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     3601 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3661 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3721 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3781 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3841 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3901 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3961 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4021 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4081 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4141 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4201 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4261 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4321 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4381 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4441 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4501 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtacgatt taaataggcc
     4561 tgactcacta tagggagacc ggaattccct ggcacgacag gtttcccgac tggaaagcgg
     4621 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttagggaccc cgggctttac
     4681 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     4741 gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg tcgtgactgg
     4801 taaaacccgg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagcagg
     4861 cgtaataagg aaaggatcca tgtactattt gaaaaacaca aacttttgga tgttcggttt
     4921 attctttttc ttttactttt ttatcatggg agcctacttc ccgtttttcc cgatttggct
     4981 acatgatatc aaccatatca gcaaaagtga tacgggtatt atttttgccg ctatttctct
     5041 gttctcgcta ttattccaac cgctgtttgg tctgctttct gacaaactcg gtctacgcaa
     5101 atacctgctg tggattatta ccggcatgtt agtgatgttt gcgccgttct ttatttttat
     5161 cttcgggcca ctgctgcagt acaacatttt agtaggatcg attgttggtg gtatttatct
     5221 aggctttagt tttaacgccg gtgcgccagc agtagaggca tttattgaga aagtcagccg
     5281 gcgcagtaat ttcgaatttg gtcgcgcgcg gatgtttggc agtgttggct gggcgctggt
     5341 tgcctcgatt gtcgggatca tgttcaccat taataatcag tttgttttct ggctgggctc
     5401 tggcagttgt ctcatcctcg ccgttttact ctttttcgcc aaaacggacg cgccctcgag
     5461 tgccacggtt gccaatgcgg taggtgccaa ccattcggca tttagcctta agctggcact
     5521 ggaactgttc agacagccaa aactgtggtt tttgtcactg tatgttattg gcgtttcctc
     5581 cacctacgat gtttttgacc aacagtttgc taatttcttt acttcgttct ttgctaccgg
     5641 tgaacagggt acccgcgtat ttggctacgt aacgacaatg ggcgaattac ttaacgcctc
     5701 gattatgttc tttgcgccac tgatcattaa tcgcatcggt gggaagaatg ccctgctgct
     5761 ggctggcact attatgtctg tacgtattat tggctcatcg ttcgccacct cagcgctgga
     5821 agtggttatt ctgaaaacgc tgcatatgtt tgaagtaccg ttcctgctgg tgggctcctt
     5881 taaatatatt actagtcagt ttgaagtgcg tttttcagcg acgatttatc tggtcagttt
     5941 cagcttcttt aagcaactgg cgatgatttt tatgtctgta ctggcgggca atatgtatga
     6001 aagcataggt ttccaaggcg cttatctggt gctgggtctg gtggcgctgg gcttcacctt
     6061 aatttccgtg ttcacgctta gcggcccggg cccgctttcc ctgctgcgt
//
