LOCUS       pKDcas9_71_cerul        8538 bp DNA     circular SYN 15-NOV-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8538)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 15, 2024 from SnapGene Viewer 7.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..546
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     terminator      complement(708..735)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(751..822)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(846..2432,2433..2435,2436..4922,4923..4925,
                     4926..4952))
                     /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"
     RBS             4959..4970
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    4989..5007
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        5015..5069
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    5015..5033
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             5085..5711
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     misc_feature    5727..5858
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   5859..5894
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   5925..5960
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   5991..6026
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        6147..6225
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             6567..7361
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     terminator      7380..7474
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      complement(7567..7594)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(7643..7672)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(8083..8094)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     RBS             8349..8360
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(8364..8398)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
ORIGIN
        1 ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca
       61 gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg
      121 aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc atgacttcaa
      181 gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt
      241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac tgaacggggg
      301 gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg
      361 gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca
      421 ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg
      481 tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta
      541 tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt
      601 ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc
      661 gtagcgagtc agtgagcgag gaagcggaat atatccctag gtataaacgc agaaaggccc
      721 acccgaaggt gagccagtgt gactctagta gagagcgttc accgacaaac aacagataaa
      781 acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgg agatccttac
      841 tcgagttagt cacctcctag ctgactcaaa tcaatgcgtg tttcataaag accagtgatg
      901 gattgatgga taagagtggc atctaaaact tcttttgtag acgtatatcg tttacgatca
      961 attgttgtat caaaatattt aaaagcagcg ggagctccaa gattcgtcaa cgtaaataaa
     1021 tgaataatat tttctgcttg ttcacgtatt ggtttgtctc tatgtttgtt atatgcacta
     1081 agaactttat ctaaattggc atctgctaaa ataacacgct tagaaaattc actgatttgc
     1141 tcaataatct catctaaata atgcttatgc tgctccacaa acaattgttt ttgttcgtta
     1201 tcttctggac tacccttcaa cttttcataa tgactagcta aatataaaaa attcacatat
     1261 ttgcttggca gagccagctc atttcctttt tgtaattctc cggcactagc cagcatccgt
     1321 ttacgaccgt tttctaactc aaaaagacta tatttaggta gtttaatgat taagtctttt
     1381 ttaacttcct tatatccttt agcttctaaa aagtcaatcg gatttttttc aaaggaactt
     1441 ctttccataa ttgtgatccc tagtaactct ttaacggatt ttaacttctt cgatttccct
     1501 ttttccacct tagcaaccac taggactgaa taagctaccg ttggactatc aaaaccacca
     1561 tatttttttg gatcccagtc ttttttacga gcaataagct tgtccgaatt tctttttggt
     1621 aaaattgact ccttggagaa tccgcctgtc tgtacttctg ttttcttgac aatattgact
     1681 tggggcatgg acaatacttt gcgcactgtg gcaaaatctc gccctttatc ccagacaatt
     1741 tctccagttt ccccattagt ttcgattaga gggcgtttgc gaatctctcc atttgcaagt
     1801 gtaatttctg ttttgaagaa gttcatgata ttagagtaaa agaaatattt tgcggttgct
     1861 ttgcctattt cttgctcaga cttagcaatc attttacgaa catcataaac tttataatca
     1921 ccatagacaa actccgattc aagttttgga tatttcttaa tcaaagcagt tccaacgacg
     1981 gcatttagat acgcatcatg ggcatgatgg taattgttaa tctcacgtac tttatagaat
     2041 tggaaatctt ttcggaagtc agaaactaat ttagatttta aggtaatcac tttaacctct
     2101 cgaataagtt tatcattttc atcgtattta gtattcatgc gactatccaa aatttgtgcc
     2161 acatgcttag tgatttggcg agtttcaacc aattggcgtt tgataaaacc agctttatca
     2221 agttcactca aacctccacg ttcagctttc gttaaattat caaacttacg ttgagtgatt
     2281 aacttggcgt ttagaagttg tctccaatag tttttcatct ttttgactac ttcttcactt
     2341 ggaacgttat ccgatttacc acgattttta tcagaacgcg ttaagacctt attgtctatt
     2401 gaatcgtctt taaggaaact ttgtggaaca atggcatcga catcataatc acttaaacga
     2461 ttaatatcta attcttggtc cacatacatg tctcttccat tttggagata atagagatag
     2521 agcttttcat tttgcaattg agtattttca acaggatgct ctttaagaat ctgacttcct
     2581 aattctttga taccttcttc gattcgtttc atacgctctc gcgaattttt ctggcccttt
     2641 tgagttgtct gattttcacg tgccatttca ataacgatat tttctggctt atgccgcccc
     2701 attactttga ccaattcatc aacaactttt acagtctgta aaataccttt tttaatagca
     2761 gggctaccag ctaaatttgc aatatgttca tgtaaactat cgccttgtcc agacacttgt
     2821 gctttttgaa tgtcttcttt aaatgtcaaa ctatcatcat ggatcagctg cataaaattg
     2881 cgattggcaa aaccatctga tttcaaaaaa tctaatattg ttttgccaga ttgcttatcc
     2941 ctaataccat taatcaattt tcgagacaaa cgtccccaac cagtataacg gcgacgttta
     3001 agctgtttca tcaccttatc atcaaagagg tgagcatatg ttttaagtct ttcctcaatc
     3061 atctccctat cttcaaataa ggtcaatgtt aaaacaatat cctctaagat atcttcattt
     3121 tcttcattat ccaaaaaatc tttatcttta ataattttta gcaaatcatg gtaggtacct
     3181 aatgaagcat taaatctatc ttcaactcct gaaatttcaa cactatcaaa acattctatt
     3241 tttttgaaat aatcttcttt taattgctta acggttactt ttcgatttgt tttgaagagt
     3301 aaatcaacaa tggctttctt ctgttcacct gaaagaaatg ctggttttcg cattccttca
     3361 gtaacatatt tgacctttgt caattcgtta taaaccgtaa aatactcata aagcaaacta
     3421 tgttttggta gtactttttc atttggaaga tttttatcaa agtttgtcat gcgttcaata
     3481 aatgattgag ctgaagcacc tttatcgaca acttcttcaa aattccatgg ggtaattgtt
     3541 tcttcagact tccgagtcat ccatgcaaaa cgactattgc cacgcgccaa tggaccaaca
     3601 taataaggaa ttcgaaaagt caagattttt tcaatcttct cacgattgtc ttttaaaaat
     3661 ggataaaagt cttcttgtct tctcaaaata gcatgcagct cacccaagtg aatttgatgg
     3721 ggaatagagc cgttgtcaaa ggtccgttgc ttgcgcagca aatcttcacg atttagtttc
     3781 accaataatt cctcagtacc atccattttt tctaaaattg gtttgataaa tttataaaat
     3841 tcttcttggc tagctccccc atcaatataa cctgcatatc cgttttttga ttgatcaaaa
     3901 aagatttctt tatacttttc tggaagttgt tgtcgaacta aagcttttaa aagagtcaag
     3961 tcttgatgat gttcatcgta gcgtttaatc attgaagctg ataggggagc cttagttatt
     4021 tcagtattta ctcttaggat atctgaaagt aaaatagcat ctgataaatt cttagctgcc
     4081 aaaaacaaat cagcatattg atctccaatt tgcgccaata aattatctaa atcatcatcg
     4141 taagtatctt ttgaaagctg taatttagca tcttctgcca aatcaaaatt tgatttaaaa
     4201 ttaggggtca aacccaatga caaagcaatg agattcccaa ataagccatt tttcttctca
     4261 ccggggagct gagcaatgag attttctaat cgtcttgatt tactcaatcg tgcagaaaga
     4321 atcgctttag catctactcc acttgcgtta atagggtttt cttcaaataa ttgattgtag
     4381 gtttgtacca actggataaa tagtttgtcc acatcactat tatcaggatt taaatctccc
     4441 tcaatcaaaa aatgaccacg aaacttaatc atatgcgcta aggccaaata gattaagcgc
     4501 aaatccgctt tatcagtaga atctaccaat ttttttcgca gatgatagat agttggatat
     4561 ttctcatgat aagcaacttc atctactata tttccaaaaa taggatgacg ttcatgcttc
     4621 ttgtcttctt ccaccaaaaa agactcttca agtcgatgaa agaaactatc atctactttc
     4681 gccatctcat ttgaaaaaat ctcctgtaga taacaaatac gattcttccg acgtgtatac
     4741 cttctacgag ctgtccgttt gagacgagtc gcttccgctg tctctccact gtcaaataaa
     4801 agagccccta taagattttt tttgatactg tggcggtctg tatttcccag aaccttgaac
     4861 tttttagacg gaaccttata ttcatcagtg atcaccgccc atccgacgct atttgtgccg
     4921 atagctaagc ctattgagta tttcttatcc atagatcctt tctcctcttt agatcttttg
     4981 aattcttttc tctatcactg atagggaggg tcaatctcta tcactgatag ggagtggtaa
     5041 aataactcta tcaacgatag agtgtcaaca aaaattagga attaatgatg tctagattag
     5101 ataaaagtaa agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt
     5161 taacaacccg taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg
     5221 taaaaaataa gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata
     5281 ctcacttttg ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa
     5341 gttttagatg tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc
     5401 ctacagaaaa acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt
     5461 tttcactaga gaatgcatta tatgcactca gcgctgtggg gcattttact ttaggttgcg
     5521 tattggaaga tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata
     5581 gtatgccgcc attattacga caagctatcg aattatttga tcaccaaggt gcagagccag
     5641 ccttcttatt cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaaa
     5701 gtgggtctta aggtaccatg ggatcctatt tcttaataac taaaaatatg gtataatact
     5761 cttaataaat gcagtaatac aggggctttt caagactgaa gtctagctga gacaaatagt
     5821 gcgattacga aattttttag acaaaaatag tctacgaggt tttagagcta tgctgttttg
     5881 aatggtccca aaaccgataa acagccctgc gttatatgag ttatgtttta gagctatgct
     5941 gttttgaatg gtcccaaaac ttgccgaaat atgcttcccg ctacctggga gttttagagc
     6001 tatgctgttt tgaatggtcc caaaacttca gcacactgag acttgttgag ttgaattcat
     6061 gagtattaag tattgtttta tggctgataa atttctttga atttctcctt gattatttgt
     6121 tataaaagtt ataaaataat cttgttggaa ccattcaaaa cagcatagca agttaaaata
     6181 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgatacttc
     6241 tattctactc tgactgcaaa ccaaaaaaac aagcgctttc aaaacgcttg ttttatcatt
     6301 tttagggaaa ttaatctctt aatcctttta tcattctaca tttaggcgct gccatcttaa
     6361 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     6421 cttagacgtc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     6481 taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agatctgatc
     6541 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     6601 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     6661 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     6721 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     6781 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     6841 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     6901 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     6961 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     7021 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     7081 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     7141 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     7201 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     7261 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     7321 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tctcgcttgg
     7381 actcctgttg atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct
     7441 cggttgccgc cgggcgtttt ttattggtga gaatccaagc actagttggt accggcatca
     7501 atagtaaaat aataaaaaag ccggattaat aatctggctt tttatattct ctctctagta
     7561 tataaacgca gaaaggccaa cccgaaggtg agccagtgtg agactatcat actcatatcg
     7621 tgtgaagatt tgctgcgcta ttcagatcct cttctgagat gagtttttgt tcgggcccaa
     7681 gcttctcact tatacaattc atccatgccg agggtaatac cggccgctgt cacaaactcc
     7741 aggagaacca tatggtcacg tttttcgttt gggtctttag agagttttga ctgcgtcgac
     7801 aggtaatggt tgtcagggag cagtaccggc ccatcgccga tcggggtatt ctgctggtag
     7861 tgatccgcca gttgcacgct cccatcttcg atattgtggc gaattttaaa gtttgccttg
     7921 atgccatttt tctgtttatc tgcagtgata taaacattat cgctaatagc gttgtattcc
     7981 agcttgtgcc caagaatgtt gccgtcttct ttgaaatcaa ttcctttcag ttcgatgcga
     8041 tttaccagcg tatcgccctc aaatttcact tccgcacggg ttttgtagtt cccatcgtcc
     8101 ttaaagaaaa ttgtgcgttc ctgaacataa ccttccggca ttgcagattt aaagaagtcg
     8161 tgttgtttca tatgatctgg gtaacgcgca aaacactgta ccccccacgt taatgtcgtg
     8221 acaagggtag gccagggcac cggcagtttg ccagtcgtac agatgaattt gagcgtgagc
     8281 ttgccgtacg tcgcgtcacc ctcgccctcg ccggtaaata attcttcgcc cttgcttacc
     8341 atttagtatt tctcctcttt agtactagta ttatacctag gactgagcta gctgtcaaag
     8401 atctttagaa ttccagaaat catccttagc gactagtaac aacttatatc gtatggggct
     8461 gacttcaggt gctacatttg aagagataaa ttgcactgaa atctagaaat attttatctg
     8521 attaataaga tgatcttc
//
