LOCUS       Exported                7287 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 7287)
  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..7287
                     /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"
     primer_bind     complement(128..147)
                     /label=DelRBTpolyA_R
     primer_bind     complement(139..168)
                     /label=VecW/OwtsgRNA_R
     misc_feature    154..168
                     /label=terminator
     misc_feature    169..265
                     /label=Nme.sgRNA-121 gRNA-I
     promoter        complement(288..536)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     promoter        567..817
                     /label=pU1a
     primer_bind     781..850
                     /label=#683-AAV- NmeCas9-F
     primer_bind     834..853
                     /label=To Amplify optimzed NmeCas9_F
     primer_bind     complement(862..882)
                     /label=AAVandSgRNA_R
     misc_feature    870..881
                     /label=Kozak
     primer_bind     complement(881..910)
                     /label=delNPNLS_AAVbckbon_R
     primer_bind     complement(883..910)
                     /label=AmpAAV1029_F
     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"
     primer_bind     complement(4188..4213)
                     /label=hNmeCas9_R
     primer_bind     complement(4190..4213)
                     /label=DelRBTpolyA_F
     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."
     promoter        4274..4522
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    4543..4639
                     /label=Nme.sgRNA-121 gRNA-II
     misc_feature    4640..4654
                     /label=terminator
     primer_bind     4659..4676
                     /label=DelRBTpolyA_R
     repeat_region   complement(4663..4803)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     promoter        5502..5606
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5607..6025
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTT"
     CDS             6026..6467
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="GGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMA
                     TTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGII
                     AALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     rep_origin      6638..7226
                     /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 taagcgcacg aatgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     4321 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     4381 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     4441 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     4501 cttgtggaaa ggacgaaaca ccggagacga ttccacgtct cagttgtagc tcccgaaacg
     4561 ttgctacaat aaggccgtct gaaaagatgt gccgcaacgc tctgcccctt aaagcttctg
     4621 ctttaagggg catcgtttat ttttttgttt aaacactacc gcaggaaccc ctagtgatgg
     4681 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     4741 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     4801 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     4861 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4921 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4981 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5041 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     5101 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5161 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     5221 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5281 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     5341 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     5401 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5461 gctcgagtta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5521 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5581 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5641 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5701 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5761 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5821 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5881 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5941 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6001 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6061 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6121 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6181 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6241 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6301 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     6361 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6421 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6481 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6541 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6601 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6661 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6721 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6781 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6841 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6901 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6961 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7021 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7081 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7141 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7201 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7261 ccttttgctg gccttttgct cacatgt
//
