LOCUS       Exported                7115 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  In vitro transcription of a mRNA encoding humanized Cas9 nuclease, 
            with 3' UTRs suitable for germline expression in C. elegans, for 
            doing CRISPR-Cas by RNA injection. Uses SP6 RNA polymerase..
ACCESSION   .
VERSION     .
KEYWORDS    SP6-hCas9-Ce-mRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7115)
  AUTHORS   Chiu H, Schwartz HT, Antoshechkin I, Sternberg PW
  TITLE     Transgene-Free Genome Editing in Caenorhabditis elegans Using 
            CRISPR-Cas.
  JOURNAL   Genetics. 2013 Aug 26.
  PUBMED    23979577
REFERENCE   2  (bases 1 to 7115)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7115
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             126..986
                     /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     complement(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1899..1916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2069..2099
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /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)."
     primer_bind     2112..2134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2131..2147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2213..2231
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2213..2230
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2277..6380
                     /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             6393..6413
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6471..6490)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6472..6490)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6497..6514)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6497..6513)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6506..6528)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(6654..7109)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6791..6812)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7003..7022
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agctcgaaat taaccctcac taaagggaac aaaagctgga gctccaccgc ggatttaggt
     2221 gacactatag aatacacgga attctagatg atccccgcgt accgagctca gaaaaaatgg
     2281 acaagaagta ctccattggg ctcgatatcg gcacaaacag cgtcggctgg gccgtcatta
     2341 cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc gatcgccaca
     2401 gcataaagaa gaacctcatt ggcgccctcc tgttcgactc cggggagacg gccgaagcca
     2461 cgcggctcaa aagaacagca cggcgcagat atacccgcag aaagaatcgg atctgctacc
     2521 tgcaggagat ctttagtaat gagatggcta aggtggatga ctctttcttc cataggctgg
     2581 aggagtcctt tttggtggag gaggataaaa agcacgagcg ccacccaatc tttggcaata
     2641 tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg aggaagaagc
     2701 ttgtagacag tactgataag gctgacttgc ggttgatcta tctcgcgctg gcgcatatga
     2761 tcaaatttcg gggacacttc ctcatcgagg gggacctgaa cccagacaac agcgatgtcg
     2821 acaaactctt tatccaactg gttcagactt acaatcagct tttcgaagag aacccgatca
     2881 acgcatccgg agttgacgcc aaagcaatcc tgagcgctag gctgtccaaa tcccggcggc
     2941 tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt ggtaatctta
     3001 tcgccctgtc actcgggctg acccccaact ttaaatctaa cttcgacctg gccgaagatg
     3061 ccaagcttca actgagcaaa gacacctacg atgatgatct cgacaatctg ctggcccaga
     3121 tcggcgacca gtacgcagac ctttttttgg cggcaaagaa cctgtcagac gccattctgc
     3181 tgagtgatat tctgcgagtg aacacggaga tcaccaaagc tccgctgagc gctagtatga
     3241 tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt gtcagacagc
     3301 aactgcctga gaagtacaag gaaattttct tcgatcagtc taaaaatggc tacgccggat
     3361 acattgacgg cggagcaagc caggaggaat tttacaaatt tattaagccc atcttggaaa
     3421 aaatggacgg caccgaggag ctgctggtaa agcttaacag agaagatctg ttgcgcaaac
     3481 agcgcacttt cgacaatgga agcatccccc accagattca cctgggcgaa ctgcacgcta
     3541 tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa aagattgaga
     3601 aaatcctcac atttcggata ccctactatg taggccccct cgcccgggga aattccagat
     3661 tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc ctggaacttc gaggaagtcg
     3721 tggataaggg ggcctctgcc cagtccttca tcgaaaggat gactaacttt gataaaaatc
     3781 tgcctaacga aaaggtgctt cctaaacact ctctgctgta cgagtacttc acagtttata
     3841 acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca ttcctgtctg
     3901 gagagcagaa gaaagctatc gtggacctcc tcttcaagac gaaccggaaa gttaccgtga
     3961 aacagctcaa agaagactat ttcaaaaaga ttgaatgttt cgactctgtt gaaatcagcg
     4021 gagtggagga tcgcttcaac gcatccctgg gaacgtatca cgatctcctg aaaatcatta
     4081 aagacaagga cttcctggac aatgaggaga acgaggacat tcttgaggac attgtcctca
     4141 cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact tacgctcatc
     4201 tcttcgacga caaagtcatg aaacagctca agaggcgccg atatacagga tgggggcggc
     4261 tgtcaagaaa actgatcaat gggatccgag acaagcagag tggaaagaca atcctggatt
     4321 ttcttaagtc cgatggattt gccaaccgga acttcatgca gttgatccat gatgactctc
     4381 tcacctttaa ggaggacatc cagaaagcac aagtttctgg ccagggggac agtcttcacg
     4441 agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg cagaccgtta
     4501 aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa gcccgagaat atcgttatcg
     4561 agatggcccg agagaaccaa actacccaga agggacagaa gaacagtagg gaaaggatga
     4621 agaggattga agagggtata aaagaactgg ggtcccaaat ccttaaggaa cacccagttg
     4681 aaaacaccca gcttcagaat gagaagctct acctgtacta cctgcagaac ggcagggaca
     4741 tgtacgtgga tcaggaactg gacatcaatc ggctctccga ctacgacgtg gatcatatcg
     4801 tgccccagtc ttttctcaaa gatgattcta ttgataataa agtgttgaca agatccgata
     4861 aaaatagagg gaagagtgat aacgtcccct cagaagaagt tgtcaagaaa atgaaaaatt
     4921 attggcggca gctgctgaac gccaaactga tcacacaacg gaagttcgat aatctgacta
     4981 aggctgaacg aggtggcctg tctgagttgg ataaagccgg cttcatcaaa aggcagcttg
     5041 ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc atgaacacca
     5101 agtacgatga aaatgacaaa ctgattcgag aggtgaaagt tattactctg aagtctaagc
     5161 tggtctcaga tttcagaaag gactttcagt tttataaggt gagagagatc aacaattacc
     5221 accatgcgca tgatgcctac ctgaatgcag tggtaggcac tgcacttatc aaaaaatatc
     5281 ccaagcttga atctgaattt gtttacggag actataaagt gtacgatgtt aggaaaatga
     5341 tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt tacagcaata
     5401 ttatgaattt tttcaagacc gagattacac tggccaatgg agagattcgg aagcgaccac
     5461 ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga caagggtagg gatttcgcga
     5521 cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt taaaaagacc gaagtacaga
     5581 ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag ctgatcgcac
     5641 gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca gtcgcttaca
     5701 gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa aaaactcaaa agcgtcaagg
     5761 aactgctggg catcacaatc atggagcgat caagcttcga aaaaaacccc atcgactttc
     5821 tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat cattaagctt cccaagtact
     5881 ctctctttga gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc gagctgcaga
     5941 aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg gccagccact
     6001 atgaaaagct caaagggtct cccgaagata atgagcagaa gcagctgttc gtggaacaac
     6061 acaaacacta ccttgatgag atcatcgagc aaataagcga attctccaaa agagtgatcc
     6121 tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg gataagccca
     6181 tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg ggcgcgcctg
     6241 cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct acaaaggagg
     6301 tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca agaatcgacc
     6361 tctctcagct cggtggagac agcagggctg accccaagaa gaagaggaag gtgtgagcct
     6421 gagctcacgt cgaccggggc cctgagatct gctgcagggt acccaattcg ccctatagtg
     6481 agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     6541 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     6601 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt
     6661 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     6721 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     6781 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     6841 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     6901 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     6961 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     7021 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     7081 cgcgcttaat gcgccgctac agggcgcgtc aggtg
//
