LOCUS       Exported                7453 bp ds-DNA     circular SYN 11-AUG-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    sa-A0A133QCR3-146aa
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"
     enhancer        154..533
                     /label=CMV enhancer
     promoter        534..737
                     /label=CMV promoter
     CDS             768..788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             768..788
                     /codon_start=1
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             810..4112
                     /codon_start=1
                     /label=sacas9-1
                     /translation="AKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE
                     GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEE
                     FSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGE
                     VRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFG
                     WKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKF
                     QIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKE
                     IIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKA
                     INLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSI
                     KVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENA
                     KYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVK
                     QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRF
                     SVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER
                     NKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEI
                     FITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKD
                     NDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKK
                     DNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLTDKGYKFITISY
                     LDVLKKDNYYYIPEQKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTR
                     NMIELDLPDIRYKEYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQ
                     LLFKRGNKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             810..3974
                     /codon_start=1
                     /label=Sa-R3
                     /translation="AKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE
                     GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEE
                     FSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGE
                     VRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFG
                     WKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKF
                     QIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKE
                     IIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKA
                     INLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSI
                     KVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENA
                     KYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVK
                     QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRF
                     SVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER
                     NKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEI
                     FITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKD
                     NDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKK
                     DNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLTDKGYKFITISY
                     LDVLKKDNYYYIPEQKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTR
                     NMIELDLPDIRYKEYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQ
                     LLFKRGN"
     primer_bind     1974..1994
                     /label=Primer 1
     primer_bind     complement(2284..2303)
                     /label=Primer 2
     primer_bind     2298..2327
                     /label=Primer 3
     primer_bind     complement(2748..2768)
                     /label=Primer 4
     primer_bind     2774..2792
                     /label=Primer 5
     misc_feature    2774
                     /label=GCCGCC
     primer_bind     3390..3407
                     /label=esa-test-F
     primer_bind     complement(3515..3536)
                     /label=SaCas9-R
     primer_bind     3522..3554
                     /label=Primer 7
     misc_feature    3537..3974
                     /label=A0A133QCR3-146aa
     primer_bind     3819..3836
                     /label=qRCR-R3PI-F
     primer_bind     complement(3957..3989)
                     /label=Primer 8
     primer_bind     complement(3957..3974)
                     /label=qRCR-R3PI-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"
     polyA_signal    4143..4350
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    4143..4350
                     /label=PolyA
     primer_bind     complement(4161..4180)
                     /label=Primer 6
     misc_feature    4358..4606
                     /label=huamn U6 promoter
                     /note="hhh"
     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
                     /gene="bla"
                     /label=AmpR promoter
     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 ccaagcggaa ctacatcctg ggcctggaca
      841 tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg
      901 ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg cggagcaaga
      961 gaggcgccag aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc
     1021 tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg
     1081 ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc
     1141 acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg
     1201 agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg
     1261 ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat
     1321 tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc
     1381 accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct
     1441 actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg
     1501 agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct
     1561 acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg
     1621 agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga
     1681 agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta
     1741 agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg
     1801 acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga
     1861 ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc
     1921 tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg
     1981 gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca
     2041 acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt
     2101 cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga
     2161 agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc
     2221 ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga
     2281 tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga
     2341 ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg
     2401 aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct
     2461 tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca
     2521 acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc ccattccagt
     2581 acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc
     2641 tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg
     2701 acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat
     2761 acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg
     2821 tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta
     2881 agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg
     2941 ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc
     3001 agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca
     3061 aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt
     3121 acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca
     3181 cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca
     3241 aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc
     3301 accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga
     3361 agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa
     3421 aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc
     3481 tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaagc
     3541 cctaccgctt cgacgtgtac ctgaccgaca agggctacaa gttcatcacc atcagctacc
     3601 tggacgtgct gaagaaggac aactactact acatccccga gcagaagtac gacaagctga
     3661 agctgggcaa ggccatcgac aagaacgcca agttcatcgc cagcttctac aagaacgacc
     3721 tgataaagct ggacggcgag atatacaaga taatcggcgt gaacagcgac acccgcaaca
     3781 tgatagagct ggacctgccc gacatccgct acaaggagta ctgcgagctg aacaacatca
     3841 agggcgagcc ccgcatcaag aagaccatcg gcaagaaggt gaacagcatc gagaagctga
     3901 ccaccgacgt gctgggcaac gtgttcacca acacccagta caccaagccc cagctgctgt
     3961 tcaagcgcgg caacaaaagg 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
//
