LOCUS       Exported                6861 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial SpCas9 expression.
ACCESSION   .
VERSION     .
KEYWORDS    pAT-9218
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(7..630)
                     /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     538..557
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        649..704
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    685..703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             717..728
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             738..803
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             810..830
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             855..4955
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4956..5003
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      5026..5097
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5113..5140
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5168..5185)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5302..5847)
                     /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(5382..5401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5961..6055
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6076..6735)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6650..6669
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6736..6838)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
       61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
      121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
      181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
      241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
      301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
      361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
      421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
      481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
      541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
      601 aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta
      661 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaaaaag
      721 aggagaaagg atctatggac tataaggacc acgacggaga ctacaaggat catgatattg
      781 attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg
      841 gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg
      901 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
      961 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     1021 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     1081 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     1141 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     1201 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     1261 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     1321 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     1381 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     1441 aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga
     1501 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     1561 tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     1621 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     1681 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     1741 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     1801 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     1861 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     1921 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     1981 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     2041 acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg
     2101 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
     2161 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
     2221 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
     2281 acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca
     2341 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
     2401 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
     2461 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
     2521 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
     2581 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
     2641 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
     2701 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
     2761 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
     2821 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
     2881 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
     2941 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
     3001 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
     3061 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
     3121 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
     3181 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
     3241 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
     3301 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
     3361 atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
     3421 tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
     3481 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
     3541 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
     3601 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
     3661 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
     3721 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
     3781 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
     3841 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
     3901 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
     3961 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
     4021 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
     4081 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
     4141 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
     4201 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
     4261 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
     4321 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
     4381 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
     4441 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
     4501 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
     4561 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
     4621 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
     4681 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
     4741 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
     4801 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
     4861 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
     4921 agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg
     4981 ccggccaggc aaaaaagaaa aagtaaggta aggatctcca ggcatcaaat aaaacgaaag
     5041 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac
     5101 tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataccta gggatatatt
     5161 ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg
     5221 cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga
     5281 gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat
     5341 ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5401 ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc
     5461 gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc
     5521 gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg
     5581 gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca
     5641 ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa
     5701 aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta
     5761 gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag
     5821 attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc
     5881 tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat
     5941 aagttgttac tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt
     6001 ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc
     6061 gagctcgata tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt
     6121 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg
     6181 catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa
     6241 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga
     6301 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca
     6361 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca
     6421 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
     6481 ccatatcacc agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag
     6541 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt
     6601 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg
     6661 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt
     6721 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     6781 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     6841 gtctcatttt cgccagatat c
//
