LOCUS       sequence-178028-        7847 bp    DNA     circular SYN 15-APR-2025
DEFINITION  pOP576 with gRNA key.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7847)
  TITLE     Tim Lu lab plasmids
REFERENCE   2  (bases 1 to 7847)
  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: pOP593
FEATURES             Location/Qualifiers
     source          1..7847
                     /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"
     promoter        4326..4360
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        4389..4464
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      complement(4477..4571)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        4598..4632
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        4661..4736
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4748..4834
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5014..5964)
                     /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      6012..6234
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(6856..7647,7648..7716))
                     /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     7479..7498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7717..7821)
                     /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 gagctttgac agctagctca gtcctaggta taatgctagc cccgaagcgc aaaatgatcc
     4381 cctgctgcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     4441 gaaaaagtgg caccgagtcg gtgctttttt gcttggattc tcaccaataa aaaacgcccg
     4501 gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat
     4561 caacaggagt ccaagcatcc ctagtcgatt agcacctttg acagctagct cagtcctagg
     4621 tataatgcta gcccccgctg tacgatgtcg cagagtatgc gttttagagc tagaaatagc
     4681 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     4741 ttggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     4801 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ccctagaagc tcgaattctc
     4861 tagagaatca ggtttgtgcc aataccagta gaaacagacg aagaatccat gggtatggac
     4921 agttttccct ttgatatgta acggtgaaca gttgttctac ttttgtttgt tagtcttgat
     4981 gcttcactga tagatacaag agccataaga acctcagatc cttccgtatt tagccagtat
     5041 gttctctagt gtggttcgtt gtttttgcgt gagccatgag aacgaaccat tgagatcata
     5101 cttactttgc atgtcactca aaaattttgc ctcaaaactg gtgagctgaa tttttgcagt
     5161 taaagcatcg tgtagtgttt ttcttagtcc gttatgtagg taggaatctg atgtaatggt
     5221 tgttggtatt ttgtcaccat tcatttttat ctggttgttc tcaagttcgg ttacgagatc
     5281 catttgtcta tctagttcaa cttggaaaat caacgtatca gtcgggcggc ctcgcttatc
     5341 aaccaccaat ttcatattgc tgtaagtgtt taaatcttta cttattggtt tcaaaaccca
     5401 ttggttaagc cttttaaact catggtagtt attttcaagc attaacatga acttaaattc
     5461 atcaaggcta atctctatat ttgccttgtg agttttcttt tgtgttagtt cttttaataa
     5521 ccactcataa atcctcatag agtatttgtt ttcaaaagac ttaacatgtt ccagattata
     5581 ttttatgaat ttttttaact ggaaaagata aggcaatatc tcttcactaa aaactaattc
     5641 taatttttcg cttgagaact tggcatagtt tgtccactgg aaaatctcaa agcctttaac
     5701 caaaggattc ctgatttcca cagttctcgt catcagctct ctggttgctt tagctaatac
     5761 accataagca ttttccctac tgatgttcat catctgaacg tattggttat aagtgaacga
     5821 taccgtccgt tctttccttg tagggttttc aatcgtgggg ttgagtagtg ccacacagca
     5881 taaaattagc ttggtttcat gctccgttaa gtcatagcga ctaatcgcta gttcatttgc
     5941 tttgaaaaca actaattcag acatacatct caattggtct aggtgatttt aatcactata
     6001 ccaattgaga tgggctagtc aatgataatt actagtcctt ttcctttgag ttgtgggtat
     6061 ctgtaaattc tgctagacct ttgctggaaa acttgtaaat tctgctagac cctctgtaaa
     6121 ttccgctaga cctttgtgtg ttttttttgt ttatattcaa gtggttataa tttatagaat
     6181 aaagaaagaa taaaaaaaga taaaaagaat agatcccagc cctgtgtata actcactact
     6241 ttagtcagtt ccgcagtatt acaaaaggat gtcgcaaacg ctgtttgctc ctctacaaaa
     6301 cagaccttaa aaccctaaag gcttaagtag caccctcgca agctcggttg cggccgcaat
     6361 cgggcaaatc gctgaatatt ccttttgtct ccgaccatca ggcacctgag tcgctgtctt
     6421 tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag tgaatggggg taaatggcac
     6481 tacaggcgcc ttttatggat tcatgcaagg aaactaccca taatacaaga aaagcccgtc
     6541 acgggcttct cagggcgttt tatggcgggt ctgctatgtg gtgctatctg actttttgct
     6601 gttcagcagt tcctgccctc tgattttcca gtctgaccac ttcggattat cccgtgacag
     6661 gtcattcaga ctggctaatg cacccagtaa ggcagcggta tcatcaacgg ggtctgacgc
     6721 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6781 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6841 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6901 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6961 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     7021 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     7081 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     7141 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     7201 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7261 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7321 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7381 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7441 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7501 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7561 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7621 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7681 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7741 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7801 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
