LOCUS       Exported                7309 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression plasmid for S. thermophilus1 Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    MSP1673
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7309)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, 
            Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
  TITLE     Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
  JOURNAL   Nature. 2015 Jun 22. doi: 10.1038/nature14592.
  PUBMED    26098369
REFERENCE   2  (bases 1 to 7309)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7309
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    3..27
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             71..3436
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus 
                     thermophilus Type II CRISPR/Cas system (Esvelt et al., 
                     2013)"
                     /label=St1Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MGSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNL
                     VRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNE
                     ELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTY
                     GQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTG
                     KRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL
                     LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRI
                     DKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFAD
                     GSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQK
                     TTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNE
                     DDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGER
                     CLYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQAL
                     DSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRV
                     VLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLN
                     LWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFS
                     YQVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKS
                     KFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNG
                     PEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADL
                     QFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSR
                     TMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGN
                     QHIIKNEGDKPKLDF"
     CDS             3449..3469
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        3538..3556
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        3618..3742
                     /label=St1 gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus 
                     thermophilus CRISPR/Cas9 system (Esvelt et al., 2013)"
     primer_bind     complement(3747..3765)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3761..3809
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4030..4689)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4604..4623
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4690..4792)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5184..5201)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5318..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(5398..5417)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6087..7169)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7131..7150
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7170..7247)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7293..3
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7293..2
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagcg acctggtgct gggcctggac atcggcatcg gcagcgtggg
      121 cgtgggcatc ctgaacaagg tgaccggcga gatcatccac aagaacagtc gcatcttccc
      181 tgctgctcag gctgagaaca acctggtgcg ccgcaccaac cgccagggtc gccggcttgc
      241 tcgccgcaag aagcaccggc gcgtgcgcct gaaccgcctg ttcgaggaga gcggcctgat
      301 caccgacttc accaagatca gcatcaacct gaacccctac cagctgcgcg tgaagggcct
      361 gaccgacgag ctgagcaacg aggagctgtt catcgccctg aagaacatgg tgaagcaccg
      421 cggcatcagc tacctggacg acgccagcga cgacggcaac agcagcgtgg gcgactacgc
      481 ccagatcgtg aaggagaaca gcaagcagct ggagaccaag acccccggcc agatccagct
      541 ggagcgctac cagacctacg gccagctgcg cggcgacttc accgtggaga aggacggcaa
      601 gaagcaccgc ctgatcaacg tgttccccac cagcgcctac cgcagcgagg ccctgcgcat
      661 cctgcagacc cagcaggagt tcaaccccca gatcaccgac gagttcatca accgctacct
      721 ggagatcctg accggcaagc gcaagtacta ccacggcccc ggcaacgaga agagccgcac
      781 cgactacggc cgctaccgca ccagcggcga gaccctggac aacatcttcg gcatcctgat
      841 cggcaagtgc accttctacc ccgacgagtt ccgcgccgcc aaggccagct acaccgccca
      901 ggagttcaat ctgctgaacg acctgaacaa cctgaccgtg cccaccgaga ccaagaagct
      961 gagcaaggag cagaagaacc agatcatcaa ctacgtgaag aacgagaagg ctatgggccc
     1021 cgccaagctg ttcaagtaca tcgccaagct gctgagctgc gacgtggccg acatcaaggg
     1081 ctaccgcatc gacaagagcg gcaaggccga gatccacacc ttcgaggcct accgcaagat
     1141 gaagaccctg gagaccctgg acatcgagca gatggaccga gagaccctgg acaagctggc
     1201 ctacgtgctg accctgaaca ccgagcgcga gggcatccag gaggccctgg agcacgagtt
     1261 cgccgacggc agcttcagcc agaaacaggt ggacgagctg gtgcagttcc gcaaggccaa
     1321 cagcagcatc ttcggcaagg gctggcacaa cttcagcgtg aagctgatga tggagctgat
     1381 ccccgagctg tacgagacca gcgaggagca gatgaccatc ctgacccgcc tgggcaagca
     1441 gaagaccacc agcagcagca acaagaccaa gtacatcgac gagaagctgc tgaccgagga
     1501 gatctacaac cccgtggtgg ccaagagcgt gcgccaggcc atcaagatcg tgaacgccgc
     1561 catcaaggag tacggcgact tcgacaacat cgtgatcgag atggcccgcg agaccaacga
     1621 ggacgacgag aagaaggcca tccagaagat ccagaaggcc aacaaggacg agaaggacgc
     1681 cgccatgctg aaggccgcca accagtacaa cggcaaggcc gagctgcccc acagcgtgtt
     1741 ccacggccac aagcagctgg ccaccaagat ccgcctgtgg caccagcagg gcgagcgctg
     1801 cctgtacacc ggcaagacca tcagcatcca cgacctgatc aacaacagca accagttcga
     1861 ggtggaccac atcctgcccc tgagcatcac cttcgacgac agcctggcca acaaggtgct
     1921 ggtgtacgcc accgccaacc aggagaaggg ccagcgcacc ccctaccagg ccctggacag
     1981 catggacgac gcctggagct tccgcgagct gaaggccttc gtgcgcgaga gcaagaccct
     2041 gagcaacaag aagaaggagt atctgctgac cgaggaggac atcagcaagt tcgacgtgcg
     2101 caagaagttc atcgagcgca acctggtgga cacccgctac gccagccgcg tggtgctgaa
     2161 cgccctgcag gagcacttcc gcgcccacaa gatcgacacc aaggtgagcg tggtgcgcgg
     2221 ccagttcacc agccagctgc gccgccactg gggcatcgag aagacccgcg acacctacca
     2281 ccaccacgcc gtggacgccc tgatcattgc ggcttctagc cagctgaacc tgtggaagaa
     2341 gcagaagaac accctggtga gctacagcga ggaccagctg ctggacatcg agaccggcga
     2401 gctgatcagc gacgacgagt acaaggagag cgtgttcaag gccccctacc agcacttcgt
     2461 ggacaccctg aagagcaagg agttcgagga cagcatcctg ttcagctacc aggtggacag
     2521 caagttcaac cgcaagatca gcgacgccac catctacgcc acccgccagg ccaaggtggg
     2581 caaggacaag gccgacgaga cctacgtgct gggcaagatc aaggacatct acacccagga
     2641 cggctacgac gccttcatga agatctacaa gaaggacaag agcaagttcc tgatgtaccg
     2701 ccacgacccc cagaccttcg agaaggtgat cgagcccatc ctggagaact accccaacaa
     2761 gcagatcaac gagaaaggca aggaggtgcc ctgcaacccc ttcctgaagt acaaggagga
     2821 gcacggctac atccgcaagt acagcaagaa gggcaacggc cccgagatca agagcctgaa
     2881 gtactacgac agcaagctgg gcaaccacat cgacatcacc cccaaggaca gcaacaacaa
     2941 ggtggtgctg cagagcgtga gcccctggcg cgccgacgtg tacttcaaca agaccaccgg
     3001 caagtacgag atcctggggc tgaagtacgc cgatctgcag tttgagaaag gcacaggcac
     3061 ctacaagatc agccaggaga agtacaacga catcaagaag aaggagggcg tggacagcga
     3121 cagcgagttc aagttcaccc tgtacaagaa cgaccttctg ctggtgaagg acaccgagac
     3181 caaggagcaa cagctgttcc gcttcctgag ccgcaccatg cccaagcaga agcactacgt
     3241 ggagctgaag ccctacgaca agcagaagtt cgagggcggc gaggccctga tcaaggtgct
     3301 gggcaacgtg gccaacagcg gccagtgcaa gaagggcctg ggcaagagca acatcagcat
     3361 ctacaaggtg cgcaccgacg tgctgggcaa ccagcacatc atcaagaacg agggcgacaa
     3421 gcccaagttg gacttcagca gggctgaccc caagaagaag aggaaggtgt gagcggccgc
     3481 ataatgctta agtcgaacag aaagtaatcg tattgtacac ggccgcataa tcgaaattaa
     3541 tacgactcac tataggactg caggtcatgc catagcgttg ttcggaacag attcaccaac
     3601 acctagtacc tgcactcgtt tttgtactct caagatttaa gtaactgtac aacgaaactt
     3661 acacagttac ttaaatcttg cagaagctac aaagataagg cttcatgccg aaatcaacac
     3721 cctgtcattt tatggcaggg tgttttccgc tgagcaataa ctagcataac ccccttgggg
     3781 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcattttga gaagcacacg
     3841 gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt
     3901 taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt ctgccattca
     3961 tccgcttatt atcacttatt caggcgtagc accaggcgtt taagggcacc aataactgcc
     4021 ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat
     4081 tctgccgaca tggaagccat cacagacggc atgatgaacc tgaatcgcca gcggcatcag
     4141 caccttgtcg ccttgcgtat aatatttgcc catagtgaaa acgggggcga agaagttgtc
     4201 catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa
     4261 aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac
     4321 atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga
     4381 tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat
     4441 caccagctca ccgtctttca ttgccatacg gaactccgga tgagcattca tcaggcgggc
     4501 aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa
     4561 ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc
     4621 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt
     4681 tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg
     4741 tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca
     4801 ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta
     4861 ttctgcgaag tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc
     4921 tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg gcttctgttt
     4981 ctatcagctg tccctcctgt tcagctactg acggggtggt gcgtaacggc aaaagcaccg
     5041 ccggacatca gcgctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt
     5101 cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg
     5161 tgatacagga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg
     5221 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata
     5281 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg
     5341 acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa
     5401 gataccaggc gtttcccctg 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 gtaattctca tgttagtcat gccccgcgcc caccggaagg
     6001 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     6061 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6121 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     6181 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     6241 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     6301 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     6361 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     6421 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     6481 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     6541 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     6601 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     6661 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     6721 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     6781 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     6841 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     6901 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     6961 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     7021 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     7081 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     7141 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     7201 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     7261 ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
//
