LOCUS       Exported                9319 bp ds-DNA     circular SYN 08-JUL-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pCas9.VRQR-sgRNA E51L65-2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9319)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, July 8, 2019 from SnapGene Viewer 3.1.2
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9319
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(185..773)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(944..1804)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1805..1909)
                     /gene="bla"
                     /note="AmpR promoter"
     promoter        1962..2202
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        2229..2304
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2462..2841
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2842..3045
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        3090..3108
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3148..3213
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /note="3xFLAG"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3220..3240
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     misc_feature    3265..7365
                     /note="Cas9/VRQR"
                     /note="For -NGA PAM (Kleinstiver, B. P. et al.  Nature 529,
                     490–495, 2016)."
     CDS             7366..7413
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7429..7482
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             7483..8187
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /note="mCherry"
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     CDS             8203..8259
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /note="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             8266..8865
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="PuroR"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    9103..9150
                     /note="HSV TK poly(A) signal"
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
       61 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      121 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      181 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      241 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      301 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      361 gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc
      421 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
      481 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
      541 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
      601 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
      661 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      721 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
      781 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
      841 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
      901 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
      961 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     1021 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     1081 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     1141 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     1201 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     1261 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     1321 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     1381 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     1441 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     1501 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     1561 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     1621 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     1681 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     1741 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     1801 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     1861 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     1921 gaaaagtgcc acctgacggt accgctagcg tcgacggatc cgagggccta tttcccatga
     1981 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     2041 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     2101 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     2161 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gggtcttcga
     2221 gaagacctgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     2281 gaaaaagtgg caccgagtcg gtgctttttt tagatctcga gtctagagga ggtcgctgag
     2341 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     2401 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     2461 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     2521 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     2581 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     2641 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     2701 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     2761 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     2821 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     2881 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     2941 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     3001 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
     3061 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
     3121 gcccgggatc caccggtcgc caccatggac tataaggacc acgacggaga ctacaaggat
     3181 catgatattg attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc
     3241 ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc
     3301 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     3361 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     3421 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     3481 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     3541 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     3601 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     3661 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     3721 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     3781 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     3841 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     3901 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     3961 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     4021 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     4081 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     4141 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     4201 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     4261 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     4321 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     4381 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     4441 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     4501 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     4561 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     4621 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     4681 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     4741 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     4801 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     4861 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     4921 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     4981 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     5041 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     5101 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     5161 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     5221 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     5281 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     5341 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     5401 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     5461 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     5521 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     5581 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     5641 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     5701 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     5761 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     5821 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     5881 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     5941 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     6001 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     6061 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     6121 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     6181 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     6241 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     6301 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     6361 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     6421 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     6481 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     6541 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     6601 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     6661 ttcgtgagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     6721 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     6781 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     6841 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     6901 ctggcctctg ccagggaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     6961 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     7021 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     7081 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     7141 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
     7201 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
     7261 aagcagtaca ggagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
     7321 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc
     7381 acgaaaaagg ccggccaggc aaaaaagaaa aaggaattcg gcagtggaga gggcagagga
     7441 agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagtgagcaa gggcgaggag
     7501 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     7561 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     7621 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     7681 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     7741 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     7801 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     7861 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa aaccatgggc
     7921 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     7981 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     8041 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     8101 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     8161 accggcggca tggacgagct gtacaaggaa ttcggaagcg gcgcaacaaa cttctcacta
     8221 ctcaaacaag caggtgacgt ggaggagaat cccgggcctc ttaagatgac cgagtacaag
     8281 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     8341 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     8401 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     8461 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     8521 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     8581 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     8641 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     8701 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     8761 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     8821 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaatcga tgcgggactc
     8881 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     8941 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     9001 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     9061 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     9121 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     9181 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     9241 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     9301 caggccctgc catagcctc
//
