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(54..677)
                     /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     585..604
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        696..751
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    732..750
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             764..775
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             785..850
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             857..877
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             902..5002
                     /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             5003..5050
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      5073..5144
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5160..5187
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5215..5232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5349..5894)
                     /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(5429..5448)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      6008..6102
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6123..6782)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6697..6716
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6783..24)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 ttggaacctc ttacgtgccg atcaacgtct cattttcgcc agatatcgac gtcttaagac
       61 ccactttcac atttaagttg tttttctaat ccgcatatga tcaattcaag gccgaataag
      121 aaggctggct ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa taatggcggc
      181 atactatcag tagtaggtgt ttccctttct tctttagcga cttgatgctc ttgatcttcc
      241 aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg catataatgc attctctagt
      301 gaaaaacctt gttggcataa aaaggctaat tgattttcga gagtttcata ctgtttttct
      361 gtaggccgtg tacctaaatg tacttttgct ccatcgcgat gacttagtaa agcacatcta
      421 aaacttttag cgttattacg taaaaaatct tgccagcttt ccccttctaa agggcaaaag
      481 tgagtatggt gcctatctaa catctcaatg gctaaggcgt cgagcaaagc ccgcttattt
      541 tttacatgcc aatacaatgt aggctgctct acacctagct tctgggcgag tttacgggtt
      601 gttaaacctt cgattccgac ctcattaagc agctctaatg cgctgttaat cactttactt
      661 ttatctaatc tagacatcat taattcctaa tttttgttga cactctatcg ttgatagagt
      721 tattttacca ctccctatca gtgatagaga aaagaattca aaaaaagagg agaaaggatc
      781 tatggactat aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga
      841 tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
      901 cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat
      961 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
     1021 cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc
     1081 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
     1141 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
     1201 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
     1261 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
     1321 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
     1381 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     1441 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     1501 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     1561 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct
     1621 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     1681 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     1741 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     1801 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     1861 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     1921 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     1981 ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     2041 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     2101 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     2161 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     2221 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     2281 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     2341 ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa
     2401 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     2461 taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
     2521 cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
     2581 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
     2641 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
     2701 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
     2761 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
     2821 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag
     2881 gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga
     2941 tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag
     3001 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
     3061 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
     3121 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
     3181 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat
     3241 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
     3301 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
     3361 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat
     3421 cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga
     3481 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
     3541 ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac
     3601 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
     3661 ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
     3721 taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
     3781 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
     3841 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
     3901 ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
     3961 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
     4021 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc
     4081 tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc
     4141 caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
     4201 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc
     4261 cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta
     4321 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa
     4381 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
     4441 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
     4501 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca
     4561 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
     4621 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
     4681 gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
     4741 cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc
     4801 catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
     4861 tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga
     4921 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga
     4981 cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa
     5041 aaagaaaaag taaggtaagg atctccaggc atcaaataaa acgaaaggct cagtcgaaag
     5101 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg
     5161 gctcaccttc gggtgggcct ttctgcgttt atacctaggg atatattccg cttcctcgct
     5221 cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg
     5281 cggagatttc ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca
     5341 aagccgtttt tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat
     5401 cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc
     5461 ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg tcattccgct gttatggccg
     5521 cgtttgtctc attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga
     5581 ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct
     5641 tgagtccaac ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt
     5701 tagaggagtt agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg
     5761 gtgactgcgc tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct
     5821 tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac
     5881 caaaacgatc tcaagaagat catcttatta atcagataaa atatttctag atttcagtgc
     5941 aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag ttgttactag
     6001 tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca
     6061 gatggagttc tgaggtcatt actggatcta tcaacaggag tccaagcgag ctcgatatca
     6121 aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     6181 acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     6241 tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg
     6301 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata
     6361 ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc
     6421 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     6481 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     6541 tcaccgtctt tcattgccat acgaaattcc ggatgagcat tcatcaggcg ggcaagaatg
     6601 tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta
     6661 atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa
     6721 tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc
     6781 attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat
     6841 cttatttcat tatggtgaaa g
//
