LOCUS       Exported                8858 bp ds-DNA     circular SYN 15-DEC-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    eSpCas9(1.1)_No_FLAG_ATP1A1_G2_Dual_sgRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8858)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Dec 15, 2016 from SnapGene 3.2.1
            http://www.snapgene.com
COMMENT     Ligation of : eSpCas9(1.1)_No_FLAG.xdna [8427  nt] : (KpnI-B 
            lunt(trimmed)[434] / XbaI[428]) to SP_gRNA_pUC19_ATP1A1_Guid 
            e_B.xdna [427  nt] : (NheI[7] / HindIII-Blunt(filled)[434])
FEATURES             Location/Qualifiers
     source          1..8858
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..249
                     /note="Human U6 Promoter"
                     /note="dnas_title=""Human U6 Promoter"" - vntifkey=""29"" -
                     label=Human U6 Promoter - 1..249"
     misc_feature    252..257
                     /note="BbsI"
                     /note="note=""direct strand 
                     translation""translation=""VF"""
     misc_feature    260..265
                     /note="BbsI"
                     /note="note=""direct strand 
                     translation""translation=""ED"""
     misc_feature    268..343
                     /note="chimeric guide RNA scaffold"
                     /note="note=""chimeric guide RNA scaffold"" - 268..343"
     misc_feature    344..349
                     /note="U6 terminator"
                     /note="note=""U6 terminator"" - 344..349"
     promoter        489..751
                     /note="U6 promoter"
     misc_feature    752
                     /note="G"
     misc_feature    753..771
                     /note="ATP1A1 G2"
     misc_feature    772..847
                     /note="chimeric guide RNA scaffold"
                     /note="note=""chimeric guide RNA scaffold"" - 268..343"
     misc_feature    848..854
                     /note="U6 terminator"
     misc_feature    861..1659
                     /note="CBh Promoter"
                     /note="note=""CBh"" - 440..1238"
     misc_feature    1666..1675
                     /note="Kozak"
                     /note="note=""direct strand 
                     translation""translation=""ATMX"""
     misc_feature    1678..1698
                     /note="SV40 NLS"
                     /note="note=""direct strand 
                     translation""translation=""PKKKRKV"""
     misc_feature    1723..5823
                     /note="eSpCas9 (1.1) ORF"
     misc_feature    4261..4263
                     /note="K848A"
                     /note="note=""direct strand translation""translation=""A"""
     misc_feature    4726..4728
                     /note="K1003A"
                     /note="note=""direct strand translation""translation=""A"""
     misc_feature    4897..4899
                     /note="R1060A"
                     /note="note=""direct strand translation""translation=""A"""
     misc_feature    5824..5871
                     /note="Nucleoplasmin NLS"
     misc_feature    5881..6112
                     /note="bGH polyA"
                     /note="note=""bGH polyA"" - 5529..5760"
     misc_feature    6121..6261
                     /note="R-ITR"
                     /note="dnas_title=""R-ITR"" - vntifkey=""21"" - label=R-ITR
                     - 5769..5909"
     rep_origin      6353..6659
                     /direction=RIGHT
                     /note="f1 Origin"
                     /note="dnas_title=""f1 Origin"" - vntifkey=""33"" - 
                     label=f1 Origin - 6001..6307"
     misc_feature    complement(7113..7177)
                     /note="bla\promoter"
                     /note="vntifkey=""21"" - label=blapromoter - 2487..2551"
     misc_feature    7178..8035
                     /note="AmpR"
                     /note="dnas_title=""AmpR"" - vntifkey=""21"" - label=AmpR -
                     6826..7683"
     rep_origin      8186..8853
                     /direction=RIGHT
                     /note="pUC ori"
                     /note="dnas_title=""pUC ori"" - vntifkey=""33"" - label=pUC
                     ori - 7834..8501"
     misc_feature    complement(8210..8797)
                     /note="Ori"
                     /note="vntifkey=""21"" - label=Ori - 867..1454"
     rep_origin      complement(8797)
                     /direction=LEFT
                     /note="RNaseH cleavage point _ORI"
                     /note="vntifkey=""33"" - label=ORI - note="" RNaseH 
                     cleavage point "" - 867..867"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agctgtacaa aaaagcaggc tttaaaggaa ccaattcagt cgactggatc
      481 cggtaccaag gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat
      541 acgatacaag gctgttagag agataattag aattaatttg actgtaaaca caaagatatt
      601 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      661 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      721 tttatatatc ttgtggaaag gacgaaacac cgatccaagc tgctacagaa ggttttagag
      781 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      841 tcggtgcttt ttttaagctc cgttacataa cttacggtaa atggcccgcc tggctgaccg
      901 cccaacgacc cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt
      961 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     1021 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag
     1081 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     1141 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
     1201 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
     1261 gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
     1321 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
     1381 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgacg
     1441 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1501 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
     1561 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
     1621 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggcccca
     1681 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc
     1741 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     1801 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     1861 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
     1921 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     1981 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     2041 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     2101 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     2161 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     2221 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     2281 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     2341 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     2401 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
     2461 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     2521 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     2581 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     2641 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     2701 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     2761 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     2821 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     2881 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     2941 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     3001 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     3061 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     3121 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
     3181 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     3241 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     3301 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     3361 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     3421 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     3481 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     3541 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     3601 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     3661 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     3721 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     3781 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     3841 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     3901 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     3961 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     4021 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     4081 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     4141 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     4201 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     4261 gccgacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     4321 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     4381 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     4441 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     4501 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     4561 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     4621 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     4681 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctgcgct ggaaagcgag
     4741 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     4801 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     4861 accgagatta ccctggccaa cggcgagatc cggaaggcgc ctctgatcga gacaaacggc
     4921 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     4981 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     5041 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     5101 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     5161 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     5221 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     5281 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     5341 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     5401 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     5461 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     5521 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     5581 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     5641 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     5701 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     5761 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     5821 gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc
     5881 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     5941 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     6001 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     6061 ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc
     6121 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     6181 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     6241 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     6301 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     6361 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     6421 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     6481 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     6541 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     6601 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     6661 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta
     6721 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     6781 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6841 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     6901 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     6961 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     7021 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     7081 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     7141 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     7201 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     7261 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     7321 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     7381 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     7441 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     7501 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     7561 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     7621 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     7681 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     7741 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     7801 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     7861 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     7921 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     7981 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     8041 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     8101 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     8161 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     8221 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     8281 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     8341 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     8401 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     8461 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     8521 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     8581 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     8641 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     8701 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     8761 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     8821 acggttcctg gccttttgct ggccttttgc tcacatgt
//
