MLM3613

LOCUS      MLM3613               7554 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses Cas9 nuclease (Streptococcus pyogenes) from CMV and T7 
            promoters.
ACCESSION   .
VERSION     .
KEYWORDS    MLM3613
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7554)
  AUTHORS   Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT,
            Yeh JR, Joung JK
  TITLE     Efficient genome editing in zebrafish using a CRISPR-Cas system.
  JOURNAL   Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2501.
  PUBMED    23360964
REFERENCE   2  (bases 1 to 7554)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7554
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             409..4512
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4519..4539
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4569..4586
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4609..4626)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4615..4839
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4910..4926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4910..4926)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4923..4945)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4934..4950
                     /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)."
     promoter        complement(4958..4988)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5003..5024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5141..5158)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5312..5900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5392..5411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6071..6931)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6694..6713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6932..7036)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7115..7134)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7306..131
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataagaaa
      421 tactcaatag gcttagatat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
      481 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
      541 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
      601 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
      661 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
      721 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
      781 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
      841 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
      901 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
      961 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
     1021 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
     1081 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
     1141 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
     1201 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
     1261 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
     1321 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
     1381 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
     1441 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
     1501 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
     1561 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
     1621 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
     1681 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
     1741 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
     1801 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
     1861 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
     1921 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
     1981 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
     2041 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
     2101 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa
     2161 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
     2221 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
     2281 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
     2341 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
     2401 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
     2461 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
     2521 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
     2581 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
     2641 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
     2701 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
     2761 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
     2821 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
     2881 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatcacat tgttccacaa
     2941 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
     3001 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
     3061 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
     3121 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
     3181 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
     3241 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
     3301 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
     3361 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
     3421 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
     3481 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
     3541 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
     3601 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
     3661 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
     3721 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
     3781 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
     3841 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
     3901 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
     3961 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
     4021 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
     4081 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
     4141 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
     4201 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
     4261 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
     4321 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
     4381 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
     4441 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
     4501 ctaggaggtg acggttctcc caagaagaag aggaaagtct cgagcggtgg agctgcagga
     4561 tgaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc
     4621 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     4681 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     4741 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     4801 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     4861 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4921 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4981 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5041 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5101 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5161 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5221 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5281 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5341 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5401 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5461 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
     5521 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5581 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5641 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5701 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     5761 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5821 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5881 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5941 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6001 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6061 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6121 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6181 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6241 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6301 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6361 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6421 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6481 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6541 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6601 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6661 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6721 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6781 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6841 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6901 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6961 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7021 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     7081 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt
     7141 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     7201 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     7261 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     7321 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     7381 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     7441 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     7501 aatgggtgga ctatttacgg taaactgccc acttggcagt acatcaagtg tatc
//