LOCUS       sequence-169626-        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: pOP587
FEATURES             Location/Qualifiers
     source          1..7847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(396..1187,1188..1256))
                     /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     1019..1038
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1257..1361)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1594..1620,1621..1623,1624..4110,4111..4113,
                     4114..5700)
                     /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        5713..5747
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        5776..5851
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      complement(5864..5958)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        5985..6019
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        6048..6123
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      6135..6221
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6401..7351)
                     /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      7399..7621
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 tgaatggggg taaatggcac tacaggcgcc ttttatggat tcatgcaagg aaactaccca
       61 taatacaaga aaagcccgtc acgggcttct cagggcgttt tatggcgggt ctgctatgtg
      121 gtgctatctg actttttgct gttcagcagt tcctgccctc tgattttcca gtctgaccac
      181 ttcggattat cccgtgacag gtcattcaga ctggctaatg cacccagtaa ggcagcggta
      241 tcatcaacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      301 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      361 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
      421 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
      481 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
      541 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
      601 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
      661 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
      721 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      781 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      841 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      901 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      961 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1021 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1081 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1141 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1201 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1261 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1321 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     1381 ccacctgcat cgatttagca cagctaacac cacgtcgtcc ctatctgctg ccctaggtct
     1441 atgagtggtt gctggataac tttacgggca tgcataaggc tcgtataata tattcaggga
     1501 gaccacaacg gtttccctct acaaataatt ttgtttaact ttgcatgcca gttgtagatc
     1561 agcatagcat agacaaccaa ggaggtagtg cacatggata agaaatactc tattggtttg
     1621 gctatcggta caaactctgt tggttgggct gttattactg atgaatacaa ggttccatcc
     1681 aagaagttca aggttttggg taacactgat agacactcca tcaaaaagaa cttgattggt
     1741 gccttgttgt tcgattctgg tgaaactgct gaagctacta gattgaaaag aaccgctaga
     1801 agaagataca ccagaagaaa gaacagaatc tgctacttgc aagaaatctt ctccaacgaa
     1861 atggccaagg ttgatgattc attcttccac agattggaag aatccttctt ggtcgaagaa
     1921 gataagaagc acgaaagaca tccaatcttc ggtaacatcg ttgatgaagt tgcttaccac
     1981 gaaaagtacc caactatcta ccatttgaga aagaagttgg ttgactctac cgataaggct
     2041 gatttgagat tgatctattt ggctttggcc cacatgatta agttcagagg tcatttcttg
     2101 atcgaaggtg atttgaaccc agataactcc gatgttgata agttgttcat ccaattagtc
     2161 caaacctaca atcaattatt cgaagaaaac ccaatcaacg cctctggtgt tgatgctaaa
     2221 gctattttgt ctgccagatt gtccaagtcc agaagattag aaaatttgat cgcccaatta
     2281 ccaggtgaaa agaagaatgg tttgttcggt aatttgattg ccttgtcttt gggtttgact
     2341 ccaaacttca agtccaattt cgatttggct gaagatgcca agttgcaatt atctaaggat
     2401 acctacgatg acgatttgga taacttgttg gctcaaatcg gtgatcaata cgctgatttg
     2461 tttttggctg ctaagaactt gtccgatgcc attttgttgt ccgatatttt gagagtcaac
     2521 accgaaatta ctaaggctcc attgtctgcc tctatgatca aaagatacga tgaacaccac
     2581 caagacttga ctttgttgaa ggctttggtc agacaacaat tacctgaaaa gtacaaagaa
     2641 attttcttcg atcaatccaa gaacggttac gccggttata ttgatggtgg tgcttctcaa
     2701 gaagaatttt acaagttcat caagccaatc ttggaaaaga tggacggtac tgaagaatta
     2761 ttggtcaagt tgaacagaga agatttgttg agaaagcaaa gaaccttcga caacggttct
     2821 attccacatc aaattcactt gggtgaattg cacgcaattt tgagaagaca agaagatttt
     2881 tatccattct tgaaggacaa cagagaaaag atcgaaaaga ttctgacctt cagaatccct
     2941 tactacgttg gtccattggc tagaggtaat tcaagatttg cctggatgac tagaaagtcc
     3001 gaagaaacta ttactccttg gaacttcgaa gaagttgtag ataagggtgc ttctgcccaa
     3061 tcctttattg aaagaatgac caacttcgac aagaacttgc caaacgaaaa ggttttgcca
     3121 aagcactctt tgttgtacga atacttcacc gtctacaacg aattgactaa ggttaagtac
     3181 gtcaccgaag gtatgagaaa accagctttt ttatccggtg aacaaaagaa ggctatcgtc
     3241 gatttgttgt tcaagaccaa cagaaaggtt actgtcaagc aattaaaaga agattacttc
     3301 aagaaaatcg aatgcttcga ctccgttgaa atttctggtg tcgaagatag attcaatgcc
     3361 tctttaggta cttaccatga cttgttgaaa atcatcaagg acaaggattt cttggacaac
     3421 gaagaaaacg aagatatttt ggaagatatt gtcttgacat tgaccttgtt tgaagataga
     3481 gaaatgattg aagaaagatt gaaaacctac gcccacttgt tcgatgataa ggttatgaag
     3541 caattaaaga gaagaagata cactggttgg ggtagattgt ccagaaaatt gattaacggt
     3601 atcagagaca agcaatccgg taagaccatt ttggactttt tgaagtctga tggtttcgct
     3661 aacagaaact tcatgcaatt aatccacgac gattccttga ctttcaaaga agatatacaa
     3721 aaggcccaag tctctggtca aggtgattct ttacatgaac atatcgctaa cttggctggt
     3781 tctccagcta ttaagaaggg tattttacaa accgttaagg tcgttgacga attggtcaaa
     3841 gttatgggta gacataagcc agaaaacatc gttatcgaaa tggctagaga aaatcaaacc
     3901 acccaaaagg gtcaaaagaa ctccagagaa agaatgaaga gaatcgaaga aggtatcaaa
     3961 gaattgggtt cccaaatttt gaaagaacac ccagttgaaa acacccaatt acaaaacgaa
     4021 aagttgtact tgtactactt gcaaaacggt agagatatgt acgttgacca agaattggac
     4081 atcaacagat tgtctgatta cgatgttgac gctatcgttc cacaatcttt tttgaaggat
     4141 gactccattg acaacaaggt cttgactaga tccgataaga atagaggtaa gtccgataac
     4201 gttccatctg aagaagtcgt taagaaaatg aagaactatt ggagacaatt attgaacgcc
     4261 aagttgatca cccaaagaaa gtttgacaat ttgaccaagg ctgaaagagg tggtttgtct
     4321 gaattggata aggcaggttt catcaaaaga caattagtag aaaccagaca aatcaccaag
     4381 cacgttgctc aaattttgga tagtagaatg aacactaagt acgacgaaaa cgacaaattg
     4441 atcagagaag ttaaggtcat taccttgaag tccaagttgg tttccgattt cagaaaggac
     4501 ttccaattct acaaggtcag agaaatcaac aactaccatc atgcacatga tgcttacttg
     4561 aatgctgttg ttggtactgc cttgattaag aagtatccaa agttggaatc cgaatttgtc
     4621 tacggtgatt acaaggttta cgacgttaga aagatgatcg ccaagtccga acaagaaatt
     4681 ggtaaagcta ctgccaaata cttcttctac tccaatatta tgaatttctt taagaccgaa
     4741 atcactttgg ccaacggtga aattagaaaa agaccattga ttgaaactaa tggtgaaaca
     4801 ggtgaaatcg tttgggataa gggtagagat tttgccactg ttagaaaggt attgtccatg
     4861 ccacaagtaa acatcgtcaa aaagaccgaa gttcaaactg gtggtttctc caaagaatcc
     4921 attttgccta agagaaactc cgataagttg atcgctagaa aaaaagactg ggacccaaaa
     4981 aagtacggtg gttttgattc tccaactgtt gcttactctg ttttggttgt tgctaaggtc
     5041 gaaaagggta agagtaagaa gttgaagtcc gtcaaagaat tattaggtat cactatcatg
     5101 gaaagatcct cattcgaaaa gaatcctatc gactttttgg aagccaaggg ttacaaagaa
     5161 gtcaagaagg acttgatcat taagttgcca aagtacagtt tgttcgaatt ggaaaatggt
     5221 agaaagagaa tgttggcttc tgccggtgaa ttacaaaagg gtaatgaatt ggctttgcca
     5281 tccaagtacg ttaatttctt atacttggcc tcccactacg aaaaattgaa aggttctcct
     5341 gaagataacg aacaaaagca attatttgtc gaacaacaca agcactactt ggacgaaatc
     5401 attgaacaaa tttccgaatt ttccaaaaga gtcattttgg ctgacgccaa tttggacaaa
     5461 gttttgtcag cttacaacaa gcacagagat aagccaatta gagaacaagc tgaaaacatc
     5521 attcacttgt tcactttgac taacttgggt gctccagctg cttttaagta tttcgatacc
     5581 actatcgaca gaaagagata cacctctacc aaagaagttt tggacgctac tttgatccac
     5641 caatctatta ctggtttgta cgaaactaga atcgacttgt ctcaattagg tggtgattaa
     5701 caagcttgag ctttgacagc tagctcagtc ctaggtataa tgctagcccc gaagcgcaaa
     5761 atgatcccct gctgcgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     5821 tcaacttgaa aaagtggcac cgagtcggtg cttttttgct tggattctca ccaataaaaa
     5881 acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg
     5941 gatctatcaa caggagtcca agcatcccta gtcgattagc acctttgaca gctagctcag
     6001 tcctaggtat aatgctagcc ccaaaccatt aacacaagag cagcttggtt ttagagctag
     6061 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     6121 tgcttttttg gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     6181 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccgccc tagaagctcg
     6241 aattctctag agaatcaggt ttgtgccaat accagtagaa acagacgaag aatccatggg
     6301 tatggacagt tttccctttg atatgtaacg gtgaacagtt gttctacttt tgtttgttag
     6361 tcttgatgct tcactgatag atacaagagc cataagaacc tcagatcctt ccgtatttag
     6421 ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag ccatgagaac gaaccattga
     6481 gatcatactt actttgcatg tcactcaaaa attttgcctc aaaactggtg agctgaattt
     6541 ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt atgtaggtag gaatctgatg
     6601 taatggttgt tggtattttg tcaccattca tttttatctg gttgttctca agttcggtta
     6661 cgagatccat ttgtctatct agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc
     6721 gcttatcaac caccaatttc atattgctgt aagtgtttaa atctttactt attggtttca
     6781 aaacccattg gttaagcctt ttaaactcat ggtagttatt ttcaagcatt aacatgaact
     6841 taaattcatc aaggctaatc tctatatttg ccttgtgagt tttcttttgt gttagttctt
     6901 ttaataacca ctcataaatc ctcatagagt atttgttttc aaaagactta acatgttcca
     6961 gattatattt tatgaatttt tttaactgga aaagataagg caatatctct tcactaaaaa
     7021 ctaattctaa tttttcgctt gagaacttgg catagtttgt ccactggaaa atctcaaagc
     7081 ctttaaccaa aggattcctg atttccacag ttctcgtcat cagctctctg gttgctttag
     7141 ctaatacacc ataagcattt tccctactga tgttcatcat ctgaacgtat tggttataag
     7201 tgaacgatac cgtccgttct ttccttgtag ggttttcaat cgtggggttg agtagtgcca
     7261 cacagcataa aattagcttg gtttcatgct ccgttaagtc atagcgacta atcgctagtt
     7321 catttgcttt gaaaacaact aattcagaca tacatctcaa ttggtctagg tgattttaat
     7381 cactatacca attgagatgg gctagtcaat gataattact agtccttttc ctttgagttg
     7441 tgggtatctg taaattctgc tagacctttg ctggaaaact tgtaaattct gctagaccct
     7501 ctgtaaattc cgctagacct ttgtgtgttt tttttgttta tattcaagtg gttataattt
     7561 atagaataaa gaaagaataa aaaaagataa aaagaataga tcccagccct gtgtataact
     7621 cactacttta gtcagttccg cagtattaca aaaggatgtc gcaaacgctg tttgctcctc
     7681 tacaaaacag accttaaaac cctaaaggct taagtagcac cctcgcaagc tcggttgcgg
     7741 ccgcaatcgg gcaaatcgct gaatattcct tttgtctccg accatcaggc acctgagtcg
     7801 ctgtcttttt cgtgacattc agttcgctgc gctcacggct ctggcag
//
