LOCUS       Exported                6898 bp ds-DNA     circular SYN 12-AUG-2020
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6898)
  AUTHORS   Sontheimer Group
  TITLE     Direct Submission
  JOURNAL   Exported Aug 12, 2020 from SnapGene 4.1.9
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6898
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     misc_feature    154..168
                     /label=terminator
     misc_feature    169..265
                     /label=Nme.sgRNA-121
     promoter        complement(288..536)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     promoter        567..817
                     /label=pU1a
     misc_feature    870..881
                     /label=Kozak
     CDS             884..904
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    911..4156
                     /label=hNme2Cas9
     primer_bind     complement(978..997)
                     /label=SeqNme2Cas9_N-term
     primer_bind     3918..3938
                     /label=SeqNme2Cas9_C-term
     CDS             4163..4210
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    4214..4261
                     /label=ShortPolyA
                     /note="Platt et al Cell. 2014 Sep 24. pii: 
                     S0092-8674(14)01163-5. doi: 10.1016/j.cell.2014.09.014."
     repeat_region   complement(4274..4414)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     promoter        5113..5217
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5218..5636
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTT"
     CDS             5637..6078
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="RRTEGANRFFAQHGGSCNSP*SLGTGAE*SHTKRRA*HHDACSNG
                     NNVAQTINWRTTYSSFPATINRLDGGG*SCRTTSALGPSGWLVYC**IWSR*AWKPRYH
                     CSTGARW*ALPYRSYLHDGESGNYG*TK*TDR*DRCLTD*ALV"
     rep_origin      6249..6837
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggcctct agagtttaaa caaaaaaata aacgatgccc
      181 cttaaagcag aagctttaag gggcagagcg ttgcggcaca tcttttcaga cggccttatt
      241 gtagcaacgt ttcgggagct acaactgaag agctggaatg ctcttccggt gtttcgtcct
      301 ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga
      361 tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta
      421 cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattct aattatctct
      481 ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctcttcc
      541 tgcccgacct tgacgtcgac tctagaatgg aggcggtact atgtagatga gaattcagga
      601 gcaaactggg aaaagcaact gcttccaaat atttgtgatt tttacagtgt agttttggaa
      661 aaactcttag cctaccaatt cttctaagtg ttttaaaatg tgggagccag tacacatgaa
      721 gttatagagt gttttaatga ggcttaaata tttaccgtaa ctatgaaatg ctacgcatat
      781 catgctgttc aggctccgtg gccacgcaac tcatacttaa gcagacagtg gttcaaagtt
      841 tttttcttcc atttcaggtg tcgtgaacac cgccaccatg gtgcctaaga agaagagaaa
      901 ggtggaagat atggccgcct tcaagcctaa cccaatcaat tacatcctgg gactggacat
      961 cggaatcgca tccgtgggat gggctatggt ggagatcgac gaggaggaga atcctatccg
     1021 gctgatcgat ctgggcgtga gagtgtttga gagggccgag gtgccaaaga ccggcgattc
     1081 tctggctatg gcccggagac tggcacggag cgtgaggcgc ctgacacgga gaagggcaca
     1141 caggctgctg agggcacgcc ggctgctgaa gagagagggc gtgctgcagg cagcagactt
     1201 cgatgagaat ggcctgatca agagcctgcc aaacaccccc tggcagctga gagcagccgc
     1261 cctggacagg aagctgacac cactggagtg gtctgccgtg ctgctgcacc tgatcaagca
     1321 ccgcggctac ctgagccagc ggaagaacga gggagagaca gcagacaagg agctgggcgc
     1381 cctgctgaag ggagtggcca acaatgccca cgccctgcag accggcgatt tcaggacacc
     1441 tgccgagctg gccctgaata agtttgagaa ggagtccggc cacatcagaa accagagggg
     1501 cgactatagc cacaccttct cccgcaagga tctgcaggcc gagctgatcc tgctgttcga
     1561 gaagcagaag gagtttggca atccacacgt gagcggaggc ctgaaggagg gaatcgagac
     1621 cctgctgatg acacagaggc ctgccctgtc cggcgacgca gtgcagaaga tgctgggaca
     1681 ctgcaccttc gagcctgcag agccaaaggc cgccaagaac acctacacag ccgagcggtt
     1741 tatctggctg acaaagctga acaatctgag aatcctggag cagggatccg agaggccact
     1801 gaccgacaca gagagggcca ccctgatgga tgagccttac cggaagtcta agctgacata
     1861 tgcccaggcc agaaagctgc tgggcctgga ggacaccgcc ttctttaagg gcctgagata
     1921 cggcaaggat aatgccgagg cctccacact gatggagatg aaggcctatc acgccatctc
     1981 tcgcgccctg gagaaggagg gcctgaagga caagaagtcc cccctgaacc tgagctccga
     2041 gctgcaggat gagatcggca ccgccttctc tctgtttaag accgacgagg atatcacagg
     2101 ccgcctgaag gacagggtgc agcctgagat cctggaggcc ctgctgaagc acatctcttt
     2161 cgataagttt gtgcagatca gcctgaaggc cctgagaagg atcgtgccac tgatggagca
     2221 gggcaagcgg tacgacgagg cctgcgccga gatctacggc gatcactatg gcaagaagaa
     2281 cacagaggag aagatctatc tgccccctat ccctgccgac gagatcagaa atcctgtggt
     2341 gctgagggcc ctgtcccagg caagaaaagt gatcaacgga gtggtgcgcc ggtacggatc
     2401 tccagcccgg atccacatcg agaccgccag agaagtgggc aagagcttca aggaccggaa
     2461 ggagatcgag aagagacagg aggagaatcg caaggatcgg gagaaggccg ccgccaagtt
     2521 tagggagtac ttccctaact ttgtgggcga gccaaagtct aaggacatcc tgaagctgcg
     2581 cctgtacgag cagcagcacg gcaagtgtct gtatagcggc aaggagatca atctggtgcg
     2641 gctgaacgag aagggctatg tggagatcga tcacgccctg cctttctcca gaacctggga
     2701 cgattctttt aacaataagg tgctggtgct gggcagcgag aaccagaata agggcaatca
     2761 gacaccatac gagtatttca atggcaagga caactccagg gagtggcagg agttcaaggc
     2821 ccgcgtggag acctctagat ttcccaggag caagaagcag cggatcctgc tgcagaagtt
     2881 cgacgaggat ggctttaagg agtgcaacct gaatgacacc agatacgtga accggttcct
     2941 gtgccagttt gtggccgatc acatcctgct gaccggcaag ggcaagagaa gggtgttcgc
     3001 ctctaatggc cagatcacaa acctgctgag gggattttgg ggactgagga aggtgcgggc
     3061 agagaatgac agacaccacg cactggatgc agtggtggtg gcatgcagca ccgtggcaat
     3121 gcagcagaag atcacaagat tcgtgaggta taaggagatg aacgcctttg acggcaagac
     3181 catcgataag gagacaggca aggtgctgca ccagaagacc cacttccccc agccttggga
     3241 gttctttgcc caggaagtga tgatccgggt gttcggcaag ccagacggca agcctgagtt
     3301 tgaggaggcc gataccccag agaagctgag gacactgctg gcagagaagc tgtctagcag
     3361 gccagaggca gtgcacgagt acgtgacccc actgttcgtg tccagggcac ccaatcggaa
     3421 gatgtctggc gcccacaagg acacactgag aagcgccaag aggtttgtga agcacaacga
     3481 gaagatctcc gtgaagagag tgtggctgac cgagatcaag ctggccgatc tggagaacat
     3541 ggtgaattac aagaacggca gggagatcga gctgtatgag gccctgaagg caaggctgga
     3601 ggcctacgga ggaaatgcca agcaggcctt cgacccaaag gataacccct tttataagaa
     3661 gggaggacag ctggtgaagg ccgtgcgggt ggagaagacc caggagagcg gcgtgctgct
     3721 gaataagaag aacgcctaca caatcgccga caatggcgat atggtgagag tggacgtgtt
     3781 ctgtaaggtg gataagaagg gcaagaatca gtactttatc gtgcctatct atgcctggca
     3841 ggtggccgag aacatcctgc cagacatcga ttgcaagggc tacagaattg acgatagcta
     3901 tacattctgt ttttccctgc acaagtatga cctgatcgcc ttccagaagg atgagaagtc
     3961 caaggtggag tttgcctact atatcaattg cgactcctct aacggcaggt tctacctggc
     4021 ctggcacgat aagggcagca aggagcagca gtttcgcatc tccacccaga atctggtgct
     4081 gatccagaag tatcaggtga acgagctggg caaggagatc aggccatgtc ggctgaagaa
     4141 gcgcccaccc gtgcgggagg ataagcggcc cgcagcaacc aagaaggcag gacaggccaa
     4201 gaagaagaag taaaataaaa gatctttatt ttcattagat ctgtgtgttg gttttttgtg
     4261 taagcgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4321 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4381 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4441 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4501 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4561 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4621 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4681 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4741 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4801 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     4861 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4921 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     4981 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5041 tgctcccggc atccgcttac agacaagctg tgctcgagtt agacgtcagg tggcactttt
     5101 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5161 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5221 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5281 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5341 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5401 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5461 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5521 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5581 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5641 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5701 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5761 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5821 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5881 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc
     5941 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6001 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6061 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6121 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6181 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6241 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6301 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6361 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     6421 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6481 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6541 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6601 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6661 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6721 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6781 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6841 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
