LOCUS       Exported                7107 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  A CMV driven de-catalyzed SaCas9 fused to KRAB domain for inhibition
            of transcription in targeted region..
ACCESSION   .
VERSION     .
KEYWORDS    pJEP302-pAAV-CMV-dSaCas9-KRAB-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7107)
  AUTHORS   Kumar N, Stanford W, Aradhana CS, Abraham ND, Dao TJ, Thaseen S, 
            Sairavi A, Gonzalez CU, Ploski JE.
  TITLE     The Development of an AAV-Based CRISPR SaCas9 Genome Editing System 
            That Can Be Delivered to Neurons in vivo and Regulated via 
            Doxycycline and Cre-Recombinase.
  JOURNAL   Front. Mol. Neurosci. (2018) 13
REFERENCE   2  (bases 1 to 7107)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7107
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..457
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        458..661
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     612..632
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     658..682
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      748..757
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             760..780
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3961..4008
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4060..4245
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     primer_bind     complement(4312..4331)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4370..4510
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4370..4499
                     /label=AAV2 ITR
     rep_origin      4585..5040
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4672..4691)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4882..4903
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5057..5076)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5176..5198
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5236..5254)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5322..5426
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5427..6287
                     /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"
     primer_bind     complement(5645..5664)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6458..7046
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6947..6966
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtggagtt ccgcgttaca taacttacgg taaatggccc
      181 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      241 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      301 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      361 cagtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      421 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      481 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      541 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      601 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      661 tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag
      721 aagacaccgg aattcggtac caccggtgcc accatggccc caaagaagaa gcggaaggtc
      781 ggtatccacg gagtcccagc agccaagcgg aactacatcc tgggcctggc catcggcatc
      841 accagcgtgg gctacggcat catcgactac gagacacggg acgtgatcga tgccggcgtg
      901 cggctgttca aagaggccaa cgtggaaaac aacgagggca ggcggagcaa gagaggcgcc
      961 agaaggctga agcggcggag gcggcataga atccagagag tgaagaagct gctgttcgac
     1021 tacaacctgc tgaccgacca cagcgagctg agcggcatca acccctacga ggccagagtg
     1081 aagggcctga gccagaagct gagcgaggaa gagttctctg ccgccctgct gcacctggcc
     1141 aagagaagag gcgtgcacaa cgtgaacgag gtggaagagg acaccggcaa cgagctgtcc
     1201 accaaagagc agatcagccg gaacagcaag gccctggaag agaaatacgt ggccgaactg
     1261 cagctggaac ggctgaagaa agacggcgaa gtgcggggca gcatcaacag attcaagacc
     1321 agcgactacg tgaaagaagc caaacagctg ctgaaggtgc agaaggccta ccaccagctg
     1381 gaccagagct tcatcgacac ctacatcgac ctgctggaaa cccggcggac ctactatgag
     1441 ggacctggcg agggcagccc cttcggctgg aaggacatca aagaatggta cgagatgctg
     1501 atgggccact gcacctactt ccccgaggaa ctgcggagcg tgaagtacgc ctacaacgcc
     1561 gacctgtaca acgccctgaa cgacctgaac aatctcgtga tcaccaggga cgagaacgag
     1621 aagctggaat attacgagaa gttccagatc atcgagaacg tgttcaagca gaagaagaag
     1681 cccaccctga agcagatcgc caaagaaatc ctcgtgaacg aagaggatat taagggctac
     1741 agagtgacca gcaccggcaa gcccgagttc accaacctga aggtgtacca cgacatcaag
     1801 gacattaccg cccggaaaga gattattgag aacgccgagc tgctggatca gattgccaag
     1861 atcctgacca tctaccagag cagcgaggac atccaggaag aactgaccaa tctgaactcc
     1921 gagctgaccc aggaagagat cgagcagatc tctaatctga agggctatac cggcacccac
     1981 aacctgagcc tgaaggccat caacctgatc ctggacgagc tgtggcacac caacgacaac
     2041 cagatcgcta tcttcaaccg gctgaagctg gtgcccaaga aggtggacct gtcccagcag
     2101 aaagagatcc ccaccaccct ggtggacgac ttcatcctga gccccgtcgt gaagagaagc
     2161 ttcatccaga gcatcaaagt gatcaacgcc atcatcaaga agtacggcct gcccaacgac
     2221 atcattatcg agctggcccg cgagaagaac tccaaggacg cccagaaaat gatcaacgag
     2281 atgcagaagc ggaaccggca gaccaacgag cggatcgagg aaatcatccg gaccaccggc
     2341 aaagagaacg ccaagtacct gatcgagaag atcaagctgc acgacatgca ggaaggcaag
     2401 tgcctgtaca gcctggaagc catccctctg gaagatctgc tgaacaaccc cttcaactat
     2461 gaggtggacc acatcatccc cagaagcgtg tccttcgaca acagcttcaa caacaaggtg
     2521 ctcgtgaagc aggaagaagc cagcaagaag ggcaaccgga ccccattcca gtacctgagc
     2581 agcagcgaca gcaagatcag ctacgaaacc ttcaagaagc acatcctgaa tctggccaag
     2641 ggcaagggca gaatcagcaa gaccaagaaa gagtatctgc tggaagaacg ggacatcaac
     2701 aggttctccg tgcagaaaga cttcatcaac cggaacctgg tggataccag atacgccacc
     2761 agaggcctga tgaacctgct gcggagctac ttcagagtga acaacctgga cgtgaaagtg
     2821 aagtccatca atggcggctt caccagcttt ctgcggcgga agtggaagtt taagaaagag
     2881 cggaacaagg ggtacaagca ccacgccgag gacgccctga tcattgccaa cgccgatttc
     2941 atcttcaaag agtggaagaa actggacaag gccaaaaaag tgatggaaaa ccagatgttc
     3001 gaggaaaagc aggccgagag catgcccgag atcgaaaccg agcaggagta caaagagatc
     3061 ttcatcaccc cccaccagat caagcacatt aaggacttca aggactacaa gtacagccac
     3121 cgggtggaca agaagcctaa tagagagctg attaacgaca ccctgtactc cacccggaag
     3181 gacgacaagg gcaacaccct gatcgtgaac aatctgaacg gcctgtacga caaggacaat
     3241 gacaagctga aaaagctgat caacaagagc cccgaaaagc tgctgatgta ccaccacgac
     3301 ccccagacct accagaaact gaagctgatt atggaacagt acggcgacga gaagaatccc
     3361 ctgtacaagt actacgagga aaccgggaac tacctgacca agtactccaa aaaggacaac
     3421 ggccccgtga tcaagaagat taagtattac ggcaacaaac tgaacgccca tctggacatc
     3481 accgacgact accccaacag cagaaacaag gtcgtgaagc tgtccctgaa gccctacaga
     3541 ttcgacgtgt acctggacaa tggcgtgtac aagttcgtga ccgtgaagaa tctggatgtg
     3601 atcaaaaaag aaaactacta cgaagtgaat agcaagtgct atgaggaagc taagaagctg
     3661 aagaagatca gcaaccaggc cgagtttatc gcctccttct acaacaacga tctgatcaag
     3721 atcaacggcg agctgtatag agtgatcggc gtgaacaacg acctgctgaa ccggatcgaa
     3781 gtgaacatga tcgacatcac ctaccgcgag tacctggaaa acatgaacga caagaggccc
     3841 cccaggatca ttaagacaat cgcctccaag acccagagca ttaagaagta cagcacagac
     3901 attctgggca acctgtatga agtgaaatct aagaagcacc ctcagatcat caaaaagggc
     3961 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg atccggcggt
     4021 ggcggaggga tggatgctaa gtcactaact gcctggtccc ggacactggt gaccttcaag
     4081 gatgtatttg tggacttcac cagggaggag tggaagctgc tggacactgc tcagcagatc
     4141 gtgtacagaa atgtgatgct ggagaactat aagaacctgg tttccttggg ttatcagctt
     4201 actaagccag atgtgatcct ccggttggag aagggagaag agccctagtc tagacatatg
     4261 gatatctgac tagtctcgag tttattgcag cttataatgg ttacaaataa agcaatagca
     4321 tcacaaattt cacaaataaa gcattttttt cactggaccg agcggccgca ggaaccccta
     4381 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4441 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4501 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4561 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4621 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     4681 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4741 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4801 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4861 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     4921 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     4981 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     5041 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     5101 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5161 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5221 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5281 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5341 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5401 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5461 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5521 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5581 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5641 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5701 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5761 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5821 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5881 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5941 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6001 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6061 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6121 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6181 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6241 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6301 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6361 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6421 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6481 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6541 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6601 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6661 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6721 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6781 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6841 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6901 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6961 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7021 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7081 ccttttgctg gccttttgct cacatgt
//
