LOCUS       Exported                7940 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  transient expression of pcoCas9 gene in plant cells.
ACCESSION   .
VERSION     .
KEYWORDS    HBT-pcoCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7940)
  AUTHORS   Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM,
            Sheen J
  TITLE     Multiplex and homologous recombination-mediated genome editing in 
            Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.
  JOURNAL   Nat Biotechnol. 2013 Aug;31(8):688-91. doi: 10.1038/nbt.2654.
  PUBMED    23929339
REFERENCE   2  (bases 1 to 7940)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7940
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(39..55)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(39..55)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(52..74)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    63..79
                     /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(87..117)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    132..153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(270..287)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(441..1029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(521..540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1200..2060)
                     /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     1823..1842
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2061..2165)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2233..2251
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2289..2311)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2411..2430
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2624..2646
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2638..2655
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2639..2655
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2722..3231
                     /label=35SPPDK promoter
                     /note="hybrid promoter consisting of the cauliflower mosaic
                     virus 35S enhancer fused to the maize C4PPDK basal promoter
                     (Yoo et al., 2007)"
     CDS             3243..3290
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             3297..3317
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3342..4778,4968..7631)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system"
                     /label=pcoCas9
                     /note="plant codon-optimized Cas9 gene containing the 
                     potato IV2 intron"
                     /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"
     intron          4779..4967
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     CDS             7632..7679
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      7688..7940
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
ORIGIN
        1 cgatgataag ctgtcaaaca tgagaattcg taatcatggt catagctgtt tcctgtgtga
       61 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
      121 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
      181 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
      241 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
      301 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      361 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
      421 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      481 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
      541 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
      601 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
      661 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
      721 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
      781 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
      841 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
      901 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
      961 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1021 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1081 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1141 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     1201 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1261 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     1321 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     1381 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     1441 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     1501 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     1561 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     1621 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     1681 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     1741 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     1801 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     1861 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     1921 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     1981 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     2041 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2101 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2161 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     2221 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     2281 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     2341 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     2401 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     2461 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
     2521 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     2581 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     2641 aaaacgacgg ccagtgccaa gctctcgaga agcttactcc aagaatatca aagatacagt
     2701 ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct
     2761 cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg
     2821 cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga
     2881 cagtggtccc aaagatggac ccccacccac aaggagcatc gtggaaaaag aagacgttcc
     2941 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc
     3001 acaatcccac tatccttcgc cccaagcttg ggcccaagct tgggtcgcgc cccacggatg
     3061 gtataagaat aaaggcattc cgcgtgcagg attcacccgt tcgcctctca ccttttcgct
     3121 gtactctctc gccacacaca ccccctctcc agctccgttg gagctccgga cagcagcagg
     3181 cgcggggcgg tcacgtagta agcagctctc ggctccctct ccccttgctc cgtggatcca
     3241 tggattacaa ggatgatgat gataaggatt acaaggatga tgatgataag atggctccaa
     3301 agaagaagag aaaggttgga atccacggag ttccagctgc tgataagaag tactctatcg
     3361 gacttgacat cggaaccaac tctgttggat gggctgttat caccgatgag tacaaggttc
     3421 catctaagaa gttcaaggtt cttggaaaca ccgatagaca ctctatcaag aagaacctta
     3481 tcggtgctct tcttttcgat tctggagaga ccgctgaggc taccagattg aagagaaccg
     3541 ctagaagaag atacaccaga agaaagaaca gaatctgcta ccttcaggaa atcttctcta
     3601 acgagatggc taaggttgat gattctttct tccacagact tgaggagtct ttccttgttg
     3661 aggaggataa gaagcacgag agacacccaa tcttcggaaa catcgttgat gaggttgctt
     3721 accacgagaa gtacccaacc atctaccacc ttagaaagaa gttggttgat tctaccgata
     3781 aggctgatct tagacttatc taccttgctc ttgctcacat gatcaagttc agaggacact
     3841 tccttatcga gggagacctt aacccagata actctgatgt tgataagttg ttcatccagc
     3901 ttgttcagac ctacaaccag cttttcgagg agaacccaat caacgcttct ggagttgatg
     3961 ctaaggctat cctttctgct agactttcta agtctcgtag acttgagaac cttatcgctc
     4021 agcttccagg agagaagaag aacggacttt tcggaaacct tatcgctctt tctcttggac
     4081 ttaccccaaa cttcaagtct aacttcgatc ttgctgagga tgctaagttg cagctttcta
     4141 aggataccta cgatgatgat cttgataacc ttcttgctca gatcggagat cagtacgctg
     4201 atcttttcct tgctgctaag aacctttctg atgctatcct tctttctgac atccttagag
     4261 ttaacaccga gatcaccaag gctccacttt ctgcttctat gatcaagaga tacgatgagc
     4321 accaccagga tcttaccctt ttgaaggctc ttgttagaca gcagcttcca gagaagtaca
     4381 aggaaatctt cttcgatcag tctaagaacg gatacgctgg atacatcgat ggaggagctt
     4441 ctcaggagga gttctacaag ttcatcaagc caatccttga gaagatggat ggaaccgagg
     4501 agcttcttgt taagttgaac agagaggatc ttcttagaaa gcagagaacc ttcgataacg
     4561 gatctatccc acaccagatc caccttggag agcttcacgc tatccttcgt agacaggagg
     4621 atttctaccc attcttgaag gataacagag agaagatcga gaagatcctt accttcagaa
     4681 tcccatacta cgttggacca cttgctagag gaaactctcg tttcgcttgg atgaccagaa
     4741 agtctgagga gaccatcacc ccttggaact tcgaggaggt aagtttctgc ttctaccttt
     4801 gatatatata taataattat cattaattag tagtaatata atatttcaaa tatttttttc
     4861 aaaataaaag aatgtagtat atagcaattg cttttctgta gtttataagt gtgtatattt
     4921 taatttataa cttttctaat atatgaccaa aatttgttga tgtgcaggtt gttgataagg
     4981 gagcttctgc tcagtctttc atcgagagaa tgaccaactt cgataagaac cttccaaacg
     5041 agaaggttct tccaaagcac tctcttcttt acgagtactt caccgtttac aacgagctta
     5101 ccaaggttaa gtacgttacc gagggaatga gaaagccagc tttcctttct ggagagcaga
     5161 agaaggctat cgttgatctt cttttcaaga ccaacagaaa ggttaccgtt aagcagttga
     5221 aggaggatta cttcaagaag atcgagtgct tcgattctgt tgaaatctct ggagttgagg
     5281 atagattcaa cgcttctctt ggaacctacc acgatctttt gaagatcatc aaggataagg
     5341 atttccttga taacgaggag aacgaggaca tccttgagga catcgttctt acccttaccc
     5401 ttttcgagga tagagagatg atcgaggaga gactcaagac ctacgctcac cttttcgatg
     5461 ataaggttat gaagcagttg aagagaagaa gatacaccgg atggggtaga ctttctcgta
     5521 agttgatcaa cggaatcaga gataagcagt ctggaaagac catccttgat ttcttgaagt
     5581 ctgatggatt cgctaacaga aacttcatgc agcttatcca cgatgattct cttaccttca
     5641 aggaggacat ccagaaggct caggtttctg gacagggaga ttctcttcac gagcacatcg
     5701 ctaaccttgc tggatctcca gctatcaaga agggaatcct tcagaccgtt aaggttgttg
     5761 atgagcttgt taaggttatg ggtagacaca agccagagaa catcgttatc gagatggcta
     5821 gagagaacca gaccacccag aagggacaga agaactctcg tgagagaatg aagagaatcg
     5881 aggagggaat caaggagctt ggatctcaaa tcttgaagga gcacccagtt gagaacaccc
     5941 agcttcagaa cgagaagttg tacctttact accttcagaa cggaagagat atgtacgttg
     6001 atcaggagct tgacatcaac agactttctg attacgatgt tgatcacatc gttccacagt
     6061 ctttcttgaa ggatgattct atcgataaca aggttcttac ccgttctgat aagaacagag
     6121 gaaagtctga taacgttcca tctgaggagg ttgttaagaa gatgaagaac tactggagac
     6181 agcttcttaa cgctaagttg atcacccaga gaaagttcga taaccttacc aaggctgaga
     6241 gaggaggact ttctgagctt gataaggctg gattcatcaa gagacagctt gttgagacca
     6301 gacagatcac caagcacgtt gctcagatcc ttgattctcg tatgaacacc aagtacgatg
     6361 agaacgataa gttgatcaga gaggttaagg ttatcacctt gaagtctaag ttggtttctg
     6421 atttcagaaa ggatttccag ttctacaagg ttagagagat caacaactac caccacgctc
     6481 acgatgctta ccttaacgct gttgttggaa ccgctcttat caagaagtac ccaaagttgg
     6541 agtctgagtt cgtttacgga gattacaagg tttacgatgt tagaaagatg atcgctaagt
     6601 ctgagcagga gatcggaaag gctaccgcta agtacttctt ctactctaac atcatgaact
     6661 tcttcaagac cgagatcacc cttgctaacg gagagatcag aaagagacca cttatcgaga
     6721 ccaacggaga gaccggagag atcgtttggg ataagggaag agatttcgct accgttagaa
     6781 aggttctttc tatgccacag gttaacatcg ttaagaaaac cgaggttcag accggaggat
     6841 tctctaagga gtctatcctt ccaaagagaa actctgataa gttgatcgct agaaagaagg
     6901 attgggaccc aaagaagtac ggaggattcg attctccaac cgttgcttac tctgttcttg
     6961 ttgttgctaa ggttgagaag ggaaagtcta agaagttgaa gtctgttaag gagcttcttg
     7021 gaatcaccat catggagcgt tcttctttcg agaagaaccc aatcgatttc cttgaggcta
     7081 agggatacaa ggaggttaag aaggatctta tcatcaagtt gccaaagtac tctcttttcg
     7141 agcttgagaa cggaagaaag agaatgcttg cttctgctgg agagcttcag aagggaaacg
     7201 agcttgctct tccatctaag tacgttaact tcctttacct tgcttctcac tacgagaagt
     7261 tgaagggatc tccagaggat aacgagcaga agcagctttt cgttgagcag cacaagcact
     7321 accttgatga gatcatcgag caaatctctg agttctctaa gagagttatc cttgctgatg
     7381 ctaaccttga taaggttctt tctgcttaca acaagcacag agataagcca atcagagagc
     7441 aggctgagaa catcatccac cttttcaccc ttaccaacct tggtgctcca gctgctttca
     7501 agtacttcga taccaccatc gatagaaaaa gatacacctc taccaaggag gttcttgatg
     7561 ctacccttat ccaccagtct atcaccggac tttacgagac cagaatcgat ctttctcagc
     7621 ttggaggaga taagagacca gctgctacca agaaggctgg acaggctaag aagaagaagt
     7681 gactgcagat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg
     7741 tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat
     7801 gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat
     7861 ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt
     7921 gtcatctatg ttactagatc
//
