LOCUS       Exported                6861 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial SpCas9-HF1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    pAT-9251
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6861)
  AUTHORS   Talas A, Huszar K, Kulcsar PI, Varga JK, Varga E, Toth E, Welker Z, 
            Erdos G, Pach PF, Welker A, Gyorgypal Z, Tusnady GE, Welker E
  TITLE     A method for characterizing Cas9 variants via a one-million target 
            sequence library of self-targeting sgRNAs.
  JOURNAL   Nucleic Acids Res. 2021 Jan 15. pii: 6101605. doi: 
            10.1093/nar/gkaa1220.
  PUBMED    33450024
REFERENCE   2  (bases 1 to 6861)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6861
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(22..645)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     553..572
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        664..719
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    700..718
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             732..743
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             753..818
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             825..845
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             870..4970
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4971..5018
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      5041..5112
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5128..5155
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5183..5200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5317..5862)
                     /direction=LEFT
                     /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.
                     "
     primer_bind     complement(5397..5416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5976..6070
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6091..6750)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6665..6684
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6751..6853)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 ttttcgccag atatcgacgt cttaagaccc actttcacat ttaagttgtt tttctaatcc
       61 gcatatgatc aattcaaggc cgaataagaa ggctggctct gcaccttggt gatcaaataa
      121 ttcgatagct tgtcgtaata atggcggcat actatcagta gtaggtgttt ccctttcttc
      181 tttagcgact tgatgctctt gatcttccaa tacgcaacct aaagtaaaat gccccacagc
      241 gctgagtgca tataatgcat tctctagtga aaaaccttgt tggcataaaa aggctaattg
      301 attttcgaga gtttcatact gtttttctgt aggccgtgta cctaaatgta cttttgctcc
      361 atcgcgatga cttagtaaag cacatctaaa acttttagcg ttattacgta aaaaatcttg
      421 ccagctttcc ccttctaaag ggcaaaagtg agtatggtgc ctatctaaca tctcaatggc
      481 taaggcgtcg agcaaagccc gcttattttt tacatgccaa tacaatgtag gctgctctac
      541 acctagcttc tgggcgagtt tacgggttgt taaaccttcg attccgacct cattaagcag
      601 ctctaatgcg ctgttaatca ctttactttt atctaatcta gacatcatta attcctaatt
      661 tttgttgaca ctctatcgtt gatagagtta ttttaccact ccctatcagt gatagagaaa
      721 agaattcaaa aaaagaggag aaaggatcta tggactataa ggaccacgac ggagactaca
      781 aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga
      841 aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg
      901 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
      961 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     1021 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     1081 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     1141 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     1201 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     1261 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     1321 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     1381 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     1441 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     1501 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     1561 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     1621 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     1681 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     1741 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     1801 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     1861 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     1921 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     1981 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     2041 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     2101 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     2161 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     2221 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     2281 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     2341 tcgagcggat gaccgccttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     2401 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     2461 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     2521 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     2581 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     2641 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     2701 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     2761 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     2821 agcggcggag atacaccggc tggggcgcgc tgagccggaa gctgatcaac ggcatccgcg
     2881 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     2941 acttcatggc gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     3001 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctcgcc ggcagccccg
     3061 ccattaagaa gggcatcctc cagacagtga aggtggtgga cgagctggtg aaagtgatgg
     3121 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     3181 agggacagaa gaacagtcgc gagagaatga agcggatcga agagggcatc aaagagctgg
     3241 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctccagaac gagaagctgt
     3301 acctgtacta cctccagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     3361 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttcttaag gacgactcca
     3421 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     3481 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     3541 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     3601 ataaggccgg cttcatcaag agacagctgg tggaaacccg ggcgatcaca aagcacgtgg
     3661 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     3721 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     3781 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     3841 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     3901 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     3961 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     4021 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     4081 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     4141 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     4201 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggatcct aagaagtacg
     4261 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     4321 gcaagtccaa gaagcttaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     4381 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     4441 aggacctgat catcaagctg cctaagtact cgctcttcga gctggaaaac ggccggaaga
     4501 gaatgctggc ctctgccggc gaattgcaga agggaaacga actggctctt ccctccaaat
     4561 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     4621 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     4681 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     4741 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     4801 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     4861 ataggaagag gtacacgtcg accaaagagg tgctggacgc caccctgatc caccagagca
     4921 tcaccggcct gtacgaaacg cgtatcgacc tgtctcagct gggaggcgac aaaaggccgg
     4981 cggccacgaa aaaggccggc caggcaaaaa agaaaaagta aggtaaggat ctccaggcat
     5041 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     5101 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     5161 acctagggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     5221 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
     5281 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
     5341 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     5401 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
     5461 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
     5521 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
     5581 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
     5641 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
     5701 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
     5761 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
     5821 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
     5881 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     5941 agccccatac gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg
     6001 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
     6061 aacaggagtc caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta
     6121 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
     6181 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa
     6241 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     6301 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     6361 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     6421 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     6481 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg
     6541 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     6601 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     6661 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     6721 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     6781 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     6841 tacgtgccga tcaacgtctc a
//
