LOCUS       Exported                7453 bp ds-DNA     circular SYN 17-FEB-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pAAV2_ShaCas9_U6_BsaI
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7453)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Nov 18, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7453
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    39..130
                     /label=AAV2 ITR
     primer_bind     137..171
                     /label=CMV-F
     enhancer        154..533
                     /label=CMV enhancer
     promoter        534..737
                     /label=CMV promoter
     primer_bind     complement(720..755)
                     /label=CMV-R
     CDS             768..788
                     /codon_start=1
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(790..812)
                     /label=SaCas9-R2
     CDS             810..3974
                     /codon_start=1
                     /label=ShaCas9
                     /translation="AKTDYILGLDIGITSVGYGIINYNDKSIIDAGVRLFPEANVENNE
                     GRRSKRGARRLKRRRIHRLERVKQLLLDYKLLDSIDVIPQSTNPYEIRVRGLREKLTKD
                     ELVIALLHLAKRRGIHNIDVIDQEEDASNELSTKEQLSKNNLLLRDKSICEVLLERYNE
                     GKVRGEKNRFKTSDIVNEIRQILETQKEVHHLDDSFIDKYIELVETRREYYEGPGEGSP
                     YGWGADLKKWYEHLMGRCTYFPEELRSVKYAYSADLFNALNDLNNLVIQRDGLNKLEYH
                     EKYHIIENVFKQKKKPTLKQISNEIGVNPEDIKGYRITKSGKENFTEFKLYHDLKKILK
                     DQSILENIQLLDQIAEIITIYQDKESIKKELEQLTEPINEIDKESISDLAGYNGTHRLS
                     LKCINLVLEELWHTSRNQMEIFAYLNIKPRKIDLQKANKIPKDMIDEFILSPVVKRTFG
                     QAINVINKVIEKYGVPKDIIIELARENNTKDKQKFINELQKKNEKTRQRINEIIGKTGN
                     QNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYQNKV
                     LVKQTENSKKGNRTPYQYLNSGEAKLSYNQFKQHVLNLSKSKDRISKKKKEYLLEERDI
                     NKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANDMDVKVKTINGSFTNHLRKVWDFN
                     KERNHGYKHHAEDALIIANADFLFKENKKLKAANKVLEQPERKEIETKIEVNSDENYQD
                     LFVIPQQVKEIKEFRDFKYSHRVDKKPNRQLINDTLYSTRVKDDDLYIISPIKNIYSKD
                     NTDLKKHFNKNPEKFLMYQKDPKTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKT
                     NNGPIVKSIKMFGKKVGNHLDVTNQYNNSRNKLVKLSFKSYRFDVYLTDKGYKFVSITY
                     LDVLKKENYYYISEAKYDKLKLNKGIDDKAKFIGSFYYNDLIELDGEVYTVIGVNNDKN
                     NVIELNLPEIRYKEYCEINNIKGSGRLRITIGKKVNSIRKLSTDVLGNRYYQSFAKKPQ
                     LVFKKGI"
     primer_bind     3776..3794
                     /label=qRCR-ShaCas9-F
     primer_bind     complement(3916..3934)
                     /label=qRCR-ShaCas9-R
     CDS             3975..4022
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     3975..3992
                     /label=SaCas9-F
     CDS             4029..4055
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4056..4082
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4083..4109
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    4143..4350
                     /label=PolyA
     primer_bind     complement(4161..4180)
                     /label=Primer 6
     promoter        4358..4598
                     /label=U6 Promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    4627..4707
                     /label=sgRNA
     repeat_region   4716..4856
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4931..5386
                     /direction=RIGHT
                     /label=f1 ori
     promoter        5668..5772
                     /label=AmpR promoter
     CDS             5773..6633
                     /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"
     rep_origin      6804..7392
                     /direction=RIGHT
                     /label=ori
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc
      781 ggaaggtcgg tatccacgga gtcccagcag ccaagacaga ttacatcctg ggcctggata
      841 tcggcatcac cagcgtgggc tatggcatca tcaactacaa tgacaagagc atcatcgacg
      901 ccggagtgcg gctgttcccc gaggccaatg ttgaaaacaa cgagggcaga agaagcaaga
      961 gaggcgccag aaggctgaag agacggcgga ttcaccggct ggaaagagtg aagcagcttc
     1021 tgctggacta caagctgctg gacagcatcg acgtgatccc tcagtctacc aacccctacg
     1081 agatccgggt gcggggcctg agagagaagc tgaccaaaga cgagctggtg atcgcgctgc
     1141 tccatttggc taagcgccgt ggaattcaca acatcgacgt gatcgaccag gaggaggatg
     1201 ccagcaacga gctgagtaca aaggagcagc tgagcaagaa caatctgctg ctgagagata
     1261 agagcatctg tgaagtcctg ctggaacggt acaacgaggg caaggtgcgc ggagagaaaa
     1321 acagattcaa aacatctgat atcgtgaacg agatcagaca gattctggag acacagaagg
     1381 aagtgcacca cctggacgat agcttcatcg acaagtacat cgaactggtg gaaacccgaa
     1441 gagaatatta cgagggccct ggcgaaggaa gtccttacgg ctggggagcc gatctgaaga
     1501 agtggtacga gcacctgatg ggccggtgca cctacttccc tgaggaactg agatccgtga
     1561 agtacgccta ctcagccgac ctgttcaatg ccctgaacga cctgaacaac ctggttatcc
     1621 agagagacgg cctgaacaag ctggaatatc acgagaagta ccacatcatt gaaaacgtgt
     1681 tcaagcagaa aaagaaacct acactgaagc agatcagcaa cgagatcggc gtgaaccctg
     1741 aagacatcaa gggctaccgg atcaccaaat ctggcaagga gaatttcacc gagttcaagt
     1801 tgtaccacga tctcaagaag atcctgaaag accagagcat cctagaaaat atccagctgc
     1861 tggaccagat cgccgagatc atcacaatct accaggacaa ggagagcatt aaaaaagaac
     1921 tggaacagct caccgagcct ataaatgaaa tcgacaagga atctatttct gacctggctg
     1981 gatacaacgg cacccacaga ctttctctga agtgcatcaa cctggtgctg gaagaactgt
     2041 ggcacaccag cagaaatcaa atggaaatct tcgcctacct gaacatcaaa cccagaaaga
     2101 tcgacctgca gaaagctaat aaaatcccca aggacatgat cgacgaattc atcctgagcc
     2161 ctgtggtgaa gcgaacattc ggccaggcca tcaacgtgat taacaaggtg attgagaagt
     2221 acggcgttcc taaggacatc atcatcgagc tcgccagaga gaacaacacc aaggacaagc
     2281 agaaattcat taacgaactc cagaagaaaa acgagaaaac cagacagcgg atcaatgaga
     2341 tcatcggaaa gaccggcaac cagaacgcca agagactggt ggaaaagatc agactgcacg
     2401 acgagcagga gggaaaatgc ctgtacagcc tggagagcat cccactggag gacctcctca
     2461 acaaccccaa ccactacgag gtggaccaca tcatacctag aagcgtgtct tttgataaca
     2521 gttatcaaaa caaggtgctc gtcaagcaaa cagaaaacag caagaagggg aatagaacac
     2581 cttaccagta cctgaacagc ggcgaggcta agctgagcta caaccagttc aagcaacacg
     2641 tgctgaacct gagcaagagc aaggatagaa tttccaagaa gaagaaagag tacctcctgg
     2701 aggaacggga cattaacaag ttcgaggtgc agaaggaatt catcaacaga aacctggtcg
     2761 acacccgata tgccacccgc gagctgacca attacctgaa ggcctatttc agcgctaacg
     2821 atatggacgt gaaggtcaag accatcaatg gaagctttac caaccacctg cggaaggttt
     2881 gggacttcaa caaggaaaga aaccacggct acaaacacca cgccgaggat gccctgatca
     2941 tcgccaatgc cgacttcctg tttaaggaga acaagaagct gaaggccgcc aacaaagtgc
     3001 tggaacagcc tgagagaaag gaaattgaaa cgaagatcga ggtgaattcc gatgagaatt
     3061 accaagatct ctttgtgatc ccccagcagg tgaaagaaat caaggagttt agagatttta
     3121 agtacagcca cagagtggac aaaaaaccta atagacagct gatcaacgat acactgtact
     3181 ccacaagagt gaaggacgac gacctgtaca tcatatctcc tatcaagaac atctacagca
     3241 aagacaacac agatctgaag aagcacttca acaaaaaccc agagaagttc ctgatgtacc
     3301 agaaagaccc caagaccttc gagaaactgg aagttattat gaagcagtac gccaacgaga
     3361 agaatcctct ggccaaatac cacgaggaaa caggcgagta cctgacgaag tacagcaaga
     3421 ccaacaacgg gccaatcgtg aaaagcatta agatgttcgg caagaaagtg ggcaatcacc
     3481 tggatgtgac caaccagtac aacaatagcc ggaacaagct ggtgaagctg agcttcaaaa
     3541 gctacagatt cgacgtgtac ctgacagaca agggctacaa gttcgtgtcc atcacctacc
     3601 tggacgtgct gaagaaagag aattactact acatcagcga ggccaagtac gacaaactga
     3661 aactaaacaa gggcatcgat gacaaggcca agttcatcgg cagcttctac tacaacgacc
     3721 tgatcgagct ggacggcgag gtgtacaccg tgatcggcgt gaataatgac aagaacaacg
     3781 tcatcgagct caatctcccc gaaattagat acaaggaata ctgcgagatt aacaacatca
     3841 agggctccgg cagactccgg atcaccatcg gcaagaaggt gaactccatt cggaagctgt
     3901 ccaccgacgt gctcggcaac cggtactacc agtctttcgc caaaaagcct cagctcgtgt
     3961 tcaagaaggg aataaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa
     4021 agggatccta cccatacgat gttccagatt acgcttaccc atacgatgtt ccagattacg
     4081 cttacccata cgatgttcca gattacgctt aagaattcct agagctcgct gatcagcctc
     4141 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     4201 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     4261 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4321 ttgggaagag aatagcaggc atgctgggga ggtacctgag ggcctatttc ccatgattcc
     4381 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     4441 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     4501 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     4561 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggag accacggcag
     4621 gtctcagttt tagtactctg gaaacagaat ctactaaaac aaggcaaaat gccgtgttta
     4681 tctcgtcaac ttgttggcga gatttttgcg gccgcaggaa cccctagtga tggagttggc
     4741 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4801 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     4861 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4921 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4981 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5041 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5101 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     5161 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5221 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
     5281 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5341 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     5401 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5461 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5521 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5581 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5641 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5701 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5761 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5821 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5881 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5941 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     6001 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6061 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6121 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6181 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6241 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6301 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6361 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6421 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6481 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6541 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6601 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6661 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6721 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6781 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6841 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6901 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     6961 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     7021 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     7081 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7141 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7201 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7261 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7321 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7381 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7441 tttgctcaca tgt
//
