LOCUS       sequence-169772-        7321 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Temperature sensitive plasmid based on pDK46. Expresses dCas9 from 
            proD. Serves as a control for pOP587 - pOP594.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7321)
  TITLE     Tim Lu lab plasmids
REFERENCE   2  (bases 1 to 7321)
  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: pOP576
FEATURES             Location/Qualifiers
     source          1..7321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(207..233,234..236,237..2723,2724..2726,2727..4313)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(4488..5438)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      5486..5708
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(6330..7121,7122..7190))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6953..6972
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7191..7295)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catcgattta gcacagctaa caccacgtcg tccctatctg ctgccctagg tctatgagtg
       61 gttgctggat aactttacgg gcatgcataa ggctcgtata atatattcag ggagaccaca
      121 acggtttccc tctacaaata attttgttta actttgcatg ccagttgtag atcagcatag
      181 catagacaac caaggaggta gtgcacatgg ataagaaata ctctattggt ttggctatcg
      241 gtacaaactc tgttggttgg gctgttatta ctgatgaata caaggttcca tccaagaagt
      301 tcaaggtttt gggtaacact gatagacact ccatcaaaaa gaacttgatt ggtgccttgt
      361 tgttcgattc tggtgaaact gctgaagcta ctagattgaa aagaaccgct agaagaagat
      421 acaccagaag aaagaacaga atctgctact tgcaagaaat cttctccaac gaaatggcca
      481 aggttgatga ttcattcttc cacagattgg aagaatcctt cttggtcgaa gaagataaga
      541 agcacgaaag acatccaatc ttcggtaaca tcgttgatga agttgcttac cacgaaaagt
      601 acccaactat ctaccatttg agaaagaagt tggttgactc taccgataag gctgatttga
      661 gattgatcta tttggctttg gcccacatga ttaagttcag aggtcatttc ttgatcgaag
      721 gtgatttgaa cccagataac tccgatgttg ataagttgtt catccaatta gtccaaacct
      781 acaatcaatt attcgaagaa aacccaatca acgcctctgg tgttgatgct aaagctattt
      841 tgtctgccag attgtccaag tccagaagat tagaaaattt gatcgcccaa ttaccaggtg
      901 aaaagaagaa tggtttgttc ggtaatttga ttgccttgtc tttgggtttg actccaaact
      961 tcaagtccaa tttcgatttg gctgaagatg ccaagttgca attatctaag gatacctacg
     1021 atgacgattt ggataacttg ttggctcaaa tcggtgatca atacgctgat ttgtttttgg
     1081 ctgctaagaa cttgtccgat gccattttgt tgtccgatat tttgagagtc aacaccgaaa
     1141 ttactaaggc tccattgtct gcctctatga tcaaaagata cgatgaacac caccaagact
     1201 tgactttgtt gaaggctttg gtcagacaac aattacctga aaagtacaaa gaaattttct
     1261 tcgatcaatc caagaacggt tacgccggtt atattgatgg tggtgcttct caagaagaat
     1321 tttacaagtt catcaagcca atcttggaaa agatggacgg tactgaagaa ttattggtca
     1381 agttgaacag agaagatttg ttgagaaagc aaagaacctt cgacaacggt tctattccac
     1441 atcaaattca cttgggtgaa ttgcacgcaa ttttgagaag acaagaagat ttttatccat
     1501 tcttgaagga caacagagaa aagatcgaaa agattctgac cttcagaatc ccttactacg
     1561 ttggtccatt ggctagaggt aattcaagat ttgcctggat gactagaaag tccgaagaaa
     1621 ctattactcc ttggaacttc gaagaagttg tagataaggg tgcttctgcc caatccttta
     1681 ttgaaagaat gaccaacttc gacaagaact tgccaaacga aaaggttttg ccaaagcact
     1741 ctttgttgta cgaatacttc accgtctaca acgaattgac taaggttaag tacgtcaccg
     1801 aaggtatgag aaaaccagct tttttatccg gtgaacaaaa gaaggctatc gtcgatttgt
     1861 tgttcaagac caacagaaag gttactgtca agcaattaaa agaagattac ttcaagaaaa
     1921 tcgaatgctt cgactccgtt gaaatttctg gtgtcgaaga tagattcaat gcctctttag
     1981 gtacttacca tgacttgttg aaaatcatca aggacaagga tttcttggac aacgaagaaa
     2041 acgaagatat tttggaagat attgtcttga cattgacctt gtttgaagat agagaaatga
     2101 ttgaagaaag attgaaaacc tacgcccact tgttcgatga taaggttatg aagcaattaa
     2161 agagaagaag atacactggt tggggtagat tgtccagaaa attgattaac ggtatcagag
     2221 acaagcaatc cggtaagacc attttggact ttttgaagtc tgatggtttc gctaacagaa
     2281 acttcatgca attaatccac gacgattcct tgactttcaa agaagatata caaaaggccc
     2341 aagtctctgg tcaaggtgat tctttacatg aacatatcgc taacttggct ggttctccag
     2401 ctattaagaa gggtatttta caaaccgtta aggtcgttga cgaattggtc aaagttatgg
     2461 gtagacataa gccagaaaac atcgttatcg aaatggctag agaaaatcaa accacccaaa
     2521 agggtcaaaa gaactccaga gaaagaatga agagaatcga agaaggtatc aaagaattgg
     2581 gttcccaaat tttgaaagaa cacccagttg aaaacaccca attacaaaac gaaaagttgt
     2641 acttgtacta cttgcaaaac ggtagagata tgtacgttga ccaagaattg gacatcaaca
     2701 gattgtctga ttacgatgtt gacgctatcg ttccacaatc ttttttgaag gatgactcca
     2761 ttgacaacaa ggtcttgact agatccgata agaatagagg taagtccgat aacgttccat
     2821 ctgaagaagt cgttaagaaa atgaagaact attggagaca attattgaac gccaagttga
     2881 tcacccaaag aaagtttgac aatttgacca aggctgaaag aggtggtttg tctgaattgg
     2941 ataaggcagg tttcatcaaa agacaattag tagaaaccag acaaatcacc aagcacgttg
     3001 ctcaaatttt ggatagtaga atgaacacta agtacgacga aaacgacaaa ttgatcagag
     3061 aagttaaggt cattaccttg aagtccaagt tggtttccga tttcagaaag gacttccaat
     3121 tctacaaggt cagagaaatc aacaactacc atcatgcaca tgatgcttac ttgaatgctg
     3181 ttgttggtac tgccttgatt aagaagtatc caaagttgga atccgaattt gtctacggtg
     3241 attacaaggt ttacgacgtt agaaagatga tcgccaagtc cgaacaagaa attggtaaag
     3301 ctactgccaa atacttcttc tactccaata ttatgaattt ctttaagacc gaaatcactt
     3361 tggccaacgg tgaaattaga aaaagaccat tgattgaaac taatggtgaa acaggtgaaa
     3421 tcgtttggga taagggtaga gattttgcca ctgttagaaa ggtattgtcc atgccacaag
     3481 taaacatcgt caaaaagacc gaagttcaaa ctggtggttt ctccaaagaa tccattttgc
     3541 ctaagagaaa ctccgataag ttgatcgcta gaaaaaaaga ctgggaccca aaaaagtacg
     3601 gtggttttga ttctccaact gttgcttact ctgttttggt tgttgctaag gtcgaaaagg
     3661 gtaagagtaa gaagttgaag tccgtcaaag aattattagg tatcactatc atggaaagat
     3721 cctcattcga aaagaatcct atcgactttt tggaagccaa gggttacaaa gaagtcaaga
     3781 aggacttgat cattaagttg ccaaagtaca gtttgttcga attggaaaat ggtagaaaga
     3841 gaatgttggc ttctgccggt gaattacaaa agggtaatga attggctttg ccatccaagt
     3901 acgttaattt cttatacttg gcctcccact acgaaaaatt gaaaggttct cctgaagata
     3961 acgaacaaaa gcaattattt gtcgaacaac acaagcacta cttggacgaa atcattgaac
     4021 aaatttccga attttccaaa agagtcattt tggctgacgc caatttggac aaagttttgt
     4081 cagcttacaa caagcacaga gataagccaa ttagagaaca agctgaaaac atcattcact
     4141 tgttcacttt gactaacttg ggtgctccag ctgcttttaa gtatttcgat accactatcg
     4201 acagaaagag atacacctct accaaagaag ttttggacgc tactttgatc caccaatcta
     4261 ttactggttt gtacgaaact agaatcgact tgtctcaatt aggtggtgat taacaagctt
     4321 gagctcgaat tctctagaga atcaggtttg tgccaatacc agtagaaaca gacgaagaat
     4381 ccatgggtat ggacagtttt ccctttgata tgtaacggtg aacagttgtt ctacttttgt
     4441 ttgttagtct tgatgcttca ctgatagata caagagccat aagaacctca gatccttccg
     4501 tatttagcca gtatgttctc tagtgtggtt cgttgttttt gcgtgagcca tgagaacgaa
     4561 ccattgagat catacttact ttgcatgtca ctcaaaaatt ttgcctcaaa actggtgagc
     4621 tgaatttttg cagttaaagc atcgtgtagt gtttttctta gtccgttatg taggtaggaa
     4681 tctgatgtaa tggttgttgg tattttgtca ccattcattt ttatctggtt gttctcaagt
     4741 tcggttacga gatccatttg tctatctagt tcaacttgga aaatcaacgt atcagtcggg
     4801 cggcctcgct tatcaaccac caatttcata ttgctgtaag tgtttaaatc tttacttatt
     4861 ggtttcaaaa cccattggtt aagcctttta aactcatggt agttattttc aagcattaac
     4921 atgaacttaa attcatcaag gctaatctct atatttgcct tgtgagtttt cttttgtgtt
     4981 agttctttta ataaccactc ataaatcctc atagagtatt tgttttcaaa agacttaaca
     5041 tgttccagat tatattttat gaattttttt aactggaaaa gataaggcaa tatctcttca
     5101 ctaaaaacta attctaattt ttcgcttgag aacttggcat agtttgtcca ctggaaaatc
     5161 tcaaagcctt taaccaaagg attcctgatt tccacagttc tcgtcatcag ctctctggtt
     5221 gctttagcta atacaccata agcattttcc ctactgatgt tcatcatctg aacgtattgg
     5281 ttataagtga acgataccgt ccgttctttc cttgtagggt tttcaatcgt ggggttgagt
     5341 agtgccacac agcataaaat tagcttggtt tcatgctccg ttaagtcata gcgactaatc
     5401 gctagttcat ttgctttgaa aacaactaat tcagacatac atctcaattg gtctaggtga
     5461 ttttaatcac tataccaatt gagatgggct agtcaatgat aattactagt ccttttcctt
     5521 tgagttgtgg gtatctgtaa attctgctag acctttgctg gaaaacttgt aaattctgct
     5581 agaccctctg taaattccgc tagacctttg tgtgtttttt ttgtttatat tcaagtggtt
     5641 ataatttata gaataaagaa agaataaaaa aagataaaaa gaatagatcc cagccctgtg
     5701 tataactcac tactttagtc agttccgcag tattacaaaa ggatgtcgca aacgctgttt
     5761 gctcctctac aaaacagacc ttaaaaccct aaaggcttaa gtagcaccct cgcaagctcg
     5821 gttgcggccg caatcgggca aatcgctgaa tattcctttt gtctccgacc atcaggcacc
     5881 tgagtcgctg tctttttcgt gacattcagt tcgctgcgct cacggctctg gcagtgaatg
     5941 ggggtaaatg gcactacagg cgccttttat ggattcatgc aaggaaacta cccataatac
     6001 aagaaaagcc cgtcacgggc ttctcagggc gttttatggc gggtctgcta tgtggtgcta
     6061 tctgactttt tgctgttcag cagttcctgc cctctgattt tccagtctga ccacttcgga
     6121 ttatcccgtg acaggtcatt cagactggct aatgcaccca gtaaggcagc ggtatcatca
     6181 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6241 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6301 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6361 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6421 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6481 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6541 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6601 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6661 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6721 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6781 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6841 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6901 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6961 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7021 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7081 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7141 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7201 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7261 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7321 g
//
