LOCUS       Exported                7188 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of CRISPRi with U6-gRNA (Bsa1 sites).
ACCESSION   .
VERSION     .
KEYWORDS    (559) pAAV EF-1a KRAB-SadCas9 U6-gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7188)
  AUTHORS   Backstrom JR, Sheng J, Wang MC, Bernardo-Colon A, Rex TS
  TITLE     Optimization of S. aureus dCas9 and CRISPRi Elements for a Single 
            Adeno-Associated Virus that Targets an Endogenous Gene.
  JOURNAL   Mol Ther Methods Clin Dev. 2020 Sep 6;19:139-148. doi: 
            10.1016/j.omtm.2020.09.001. eCollection 2020 Dec 11.
  PUBMED    33024795
REFERENCE   2  (bases 1 to 7188)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7188
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        180..391
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             425..610
                     /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"
     CDS             3863..3889
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     polyA_signal    3933..4054
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3944..3963)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3998..4017
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4093..4333
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     4093..4113
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4264..4283
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4362..4437
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4451..4591
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4451..4580
                     /label=AAV2 ITR
     rep_origin      4666..5121
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4753..4772)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4963..4984
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5138..5157)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5257..5279
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5317..5335)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5403..5507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5508..6368
                     /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(5726..5745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6539..7127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7028..7047
                     /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 gcgttcagtc ctagtcgtga ggctccggtg cccgtcagtg
      181 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
      241 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
      301 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
      361 ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga aaactacccg aattcgccac
      421 catgcggaca ctagtgacct tcaaggatgt atttgtggac ttcaccaggg aggagtggaa
      481 gctgctggac actgctcagc agatcgtgta cagaaatgtg atgctggaga actataagaa
      541 cctggtttcc ttgggttatc agcttactaa gccagatgtg atcctccggt tggagaaggg
      601 agaagagccc ggcggttccg gcggagggtc catgggcaaa cggaactaca tcctggggct
      661 tgccattggg ataaccagcg ttggctacgg aattattgat tatgagacac gcgatgtgat
      721 tgacgccggg gttaggctgt tcaaagaggc caacgttgaa aacaacgagg gaagacggag
      781 taagcgcgga gcaagaagac tcaagcgcag acggagacat cggattcaga gggtgaaaaa
      841 gctgctcttc gattacaatc tcctgaccga tcatagtgag ctgagcggaa tcaaccccta
      901 cgaggcgcga gtgaaagggc tttcccagaa gctgtccgaa gaggagttct ccgccgcgtt
      961 gctgcacctg gccaaacgga ggggggttca caatgtaaac gaagtggagg aggacacggg
     1021 caatgaactt agtacgaaag aacagatcag taggaactct aaggctctcg aagagaaata
     1081 cgtcgctgag ttgcagcttg agagactgaa aaaagacggc gaagtacgcg gatctattaa
     1141 taggttcaag acttcagatt acgtaaagga agccaagcag ctcctgaaag tacagaaagc
     1201 gtaccatcag ctcgatcaga gcttcatcga tacctacata gatttgctgg agacacggag
     1261 gacatactac gagggcccag gggaaggatc tccttttggg tggaaggaca tcaaggaatg
     1321 gtacgagatg cttatgggac attgtacata ttttccggag gagctcagga gcgtcaagta
     1381 cgcctacaat gccgacctgt acaatgccct caatgacctc aataacctcg tgattaccag
     1441 ggacgagaac gagaagctgg agtactatga aaagttccag attatcgaga atgtgtttaa
     1501 gcagaagaag aagccgacac ttaagcagat tgcaaaggaa atcctcgtga atgaggaaga
     1561 tatcaaggga tacagagtga caagtacagg caagcccgag ttcacaaatc tgaaggtgta
     1621 ccacgatatt aaggacataa ccgcacgaaa ggagataatc gaaaacgctg agctcctcga
     1681 tcagatcgca aaaattctta ccatctacca gtctagtgag gacattcagg aggaactgac
     1741 taatctgaac agtgagctca cccaagagga aattgagcag atttcaaacc tgaaaggcta
     1801 caccgggacg cacaatctga gcctcaaagc aatcaacctc attctggatg aactttggca
     1861 cacaaatgac aaccaaattg ccatattcaa ccgcctgaaa ctggtgccaa aaaaagtgga
     1921 tctgtcacag caaaaggaaa tccctacaac cttggttgac gattttattc tgtcccccgt
     1981 tgtcaagcgg agcttcatcc agtcaatcaa ggtgatcaat gccatcatta aaaaatacgg
     2041 attgccaaac gatataatta tcgagcttgc acgagagaag aactcaaagg acgcccagaa
     2101 gatgattaac gaaatgcaga agcgcaaccg ccagacaaac gaacgcatag aggaaattat
     2161 aagaacaacc ggcaaagaga atgccaagta tctgatcgag aaaatcaagc tgcacgacat
     2221 gcaagaaggc aagtgcctgt actctctgga agctatccca ctcgaagatc tgctgaataa
     2281 tccattcaat tacgaggtgg acgccatcat ccctagatcc gtaagctttg acaattcctt
     2341 caataacaaa gttctggtta aacaggagga aaattctaaa aaagggaacc ggaccccgtt
     2401 ccagtacctg agctccagtg acagcaagat tagctacgag acttttaaga aacatattct
     2461 gaatctggcc aaaggcaaag gcaggatcag caagaccaag aaggagtacc tcctcgaaga
     2521 acgcgacatt aacagattta gtgtgcagaa agatttcatc aaccgaaacc ttgtcgatac
     2581 tcggtacgcc acgagaggcc tgatgaatct cctcaggagc tacttccgcg tcaataatct
     2641 ggacgttaaa gtcaagagca taaatggggg attcaccagc tttctgagga gaaagtggaa
     2701 gtttaagaag gaacgaaaca aaggatacaa gcaccatgct gaggatgctt tgatcatcgc
     2761 taacgcggac tttatcttta aggaatggaa aaagctggat aaggcaaaga aagtgatgga
     2821 aaaccagatg ttcgaggaga agcaggcaga gtcaatgcct gagatcgaga cagagcagga
     2881 atacaaggaa attttcatca cccctcatca gattaaacac ataaaggact tcaaagacta
     2941 taaatactct catagggtgg acaaaaaacc caatcgcgag ctcattaatg acaccctgta
     3001 ctcaacacgg aaggatgata aaggtaatac cttgattgtg aataatctta atggattgta
     3061 tgacaaagat aacgacaagc tcaagaagct gatcaacaag tctccagaga agctccttat
     3121 gtatcaccac gacccacaga cttatcagaa attgaaactg atcatggagc aatacgggga
     3181 tgagaagaac ccactctaca aatattatga ggaaacaggt aattacctga ccaagtactc
     3241 caagaaggat aacggaccag tgatcaaaaa gataaagtac tatggcaaca aacttaatgc
     3301 gcatttggac ataactgacg attaccccaa ttctcgaaac aaggttgtga agctctccct
     3361 gaagccttat agatttgacg tgtacctgga taatggggtt tataaattcg tcaccgtgaa
     3421 aaatctggac gtgatcaaaa aggagaacta ttatgaagta aactcaaagt gctatgagga
     3481 ggcgaagaag ctgaagaaga tctccaatca ggccgagttc atcgcttcct tctataataa
     3541 cgatctcatc aagatcaatg gagagcttta tcgcgtcatt ggtgtgaaca atgacttgct
     3601 gaacaggatc gaagtcaata tgatagacat tacctaccgg gagtatctcg aaaacatgaa
     3661 tgataaacgg ccgcctcgca tcatcaagac aatcgcatct aaaactcagt caataaaaaa
     3721 gtactctacc gatatcctgg ggaatctcta tgaagtgaag tcaaagaagc acccacaaat
     3781 cattaaaaaa ggtggatccg cagaagcagc agcaaaggaa gcagcagcaa aggaagcagc
     3841 agcaaaggca gtcgacaccg gtccagcagc taagagagtt aaactagatt agcggccgca
     3901 tcgtcaggcc aagcttccat cgatagacat gataagatac attgatgagt ttggacaaac
     3961 cacaacaaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     4021 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     4081 gtttcaggta ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     4141 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     4201 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     4261 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     4321 ttgtggaaag gacgaaacac cggagaccac ggcaggtctc agttttagta ctctggaaac
     4381 agaatctact aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt
     4441 ttgcggccct aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     4501 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     4561 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     4621 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     4681 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4741 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4801 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4861 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     4921 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4981 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     5041 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     5101 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     5161 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5221 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5281 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     5341 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5401 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5461 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5521 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5581 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5641 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5701 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5761 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5821 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5881 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5941 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6001 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6061 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6121 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6181 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     6241 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6301 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6361 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6421 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6481 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6541 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6601 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6661 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6721 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6781 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6841 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6901 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6961 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7021 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7081 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7141 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
