LOCUS       Exported                7284 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of CRISPRi with gSTOP gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    (565) pAAV Alb-AAT KRAB-SadCas9 U6-gSTOP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7284)
  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 7284)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7284
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             521..706
                     /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             3959..3985
                     /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    4029..4150
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4040..4059)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4094..4113
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4189..4429
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     4189..4209
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4360..4379
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4458..4533
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4547..4687
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4547..4676
                     /label=AAV2 ITR
     rep_origin      4762..5217
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4849..4868)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5059..5080
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5234..5253)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5353..5375
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5413..5431)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5499..5603
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5604..6464
                     /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(5822..5841)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6635..7223
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7124..7143
                     /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 gcgtaaataa cctgcgttat agcatccact cagtatccct
      181 tgagcatgag gtgacactac ttaacatagg gacgagatgg tactttgtgt ctcctgctct
      241 gtcagcaggg cactgtactt gctgatacag ggaatgtttg ttcttaaata ccatcattcc
      301 ggacgtgttt gccttggcag tttccatgta catgcagaaa gaagtttgga taggcgggcg
      361 actcagatcc cagccagtgg acttagcccc tgtttgctcc tccgataact ggggtgacct
      421 tggttaatat tcaccagcag cctcccccgt tgcccctctg gatccactgc ttaaatacgg
      481 acgaggacag ggccctgtct cctcagaatt cgccaccatg cggacactag tgaccttcaa
      541 ggatgtattt gtggacttca ccagggagga gtggaagctg ctggacactg ctcagcagat
      601 cgtgtacaga aatgtgatgc tggagaacta taagaacctg gtttccttgg gttatcagct
      661 tactaagcca gatgtgatcc tccggttgga gaagggagaa gagcccggcg gttccggcgg
      721 agggtccatg ggcaaacgga actacatcct ggggcttgcc attgggataa ccagcgttgg
      781 ctacggaatt attgattatg agacacgcga tgtgattgac gccggggtta ggctgttcaa
      841 agaggccaac gttgaaaaca acgagggaag acggagtaag cgcggagcaa gaagactcaa
      901 gcgcagacgg agacatcgga ttcagagggt gaaaaagctg ctcttcgatt acaatctcct
      961 gaccgatcat agtgagctga gcggaatcaa cccctacgag gcgcgagtga aagggctttc
     1021 ccagaagctg tccgaagagg agttctccgc cgcgttgctg cacctggcca aacggagggg
     1081 ggttcacaat gtaaacgaag tggaggagga cacgggcaat gaacttagta cgaaagaaca
     1141 gatcagtagg aactctaagg ctctcgaaga gaaatacgtc gctgagttgc agcttgagag
     1201 actgaaaaaa gacggcgaag tacgcggatc tattaatagg ttcaagactt cagattacgt
     1261 aaaggaagcc aagcagctcc tgaaagtaca gaaagcgtac catcagctcg atcagagctt
     1321 catcgatacc tacatagatt tgctggagac acggaggaca tactacgagg gcccagggga
     1381 aggatctcct tttgggtgga aggacatcaa ggaatggtac gagatgctta tgggacattg
     1441 tacatatttt ccggaggagc tcaggagcgt caagtacgcc tacaatgccg acctgtacaa
     1501 tgccctcaat gacctcaata acctcgtgat taccagggac gagaacgaga agctggagta
     1561 ctatgaaaag ttccagatta tcgagaatgt gtttaagcag aagaagaagc cgacacttaa
     1621 gcagattgca aaggaaatcc tcgtgaatga ggaagatatc aagggataca gagtgacaag
     1681 tacaggcaag cccgagttca caaatctgaa ggtgtaccac gatattaagg acataaccgc
     1741 acgaaaggag ataatcgaaa acgctgagct cctcgatcag atcgcaaaaa ttcttaccat
     1801 ctaccagtct agtgaggaca ttcaggagga actgactaat ctgaacagtg agctcaccca
     1861 agaggaaatt gagcagattt caaacctgaa aggctacacc gggacgcaca atctgagcct
     1921 caaagcaatc aacctcattc tggatgaact ttggcacaca aatgacaacc aaattgccat
     1981 attcaaccgc ctgaaactgg tgccaaaaaa agtggatctg tcacagcaaa aggaaatccc
     2041 tacaaccttg gttgacgatt ttattctgtc ccccgttgtc aagcggagct tcatccagtc
     2101 aatcaaggtg atcaatgcca tcattaaaaa atacggattg ccaaacgata taattatcga
     2161 gcttgcacga gagaagaact caaaggacgc ccagaagatg attaacgaaa tgcagaagcg
     2221 caaccgccag acaaacgaac gcatagagga aattataaga acaaccggca aagagaatgc
     2281 caagtatctg atcgagaaaa tcaagctgca cgacatgcaa gaaggcaagt gcctgtactc
     2341 tctggaagct atcccactcg aagatctgct gaataatcca ttcaattacg aggtggacgc
     2401 catcatccct agatccgtaa gctttgacaa ttccttcaat aacaaagttc tggttaaaca
     2461 ggaggaaaat tctaaaaaag ggaaccggac cccgttccag tacctgagct ccagtgacag
     2521 caagattagc tacgagactt ttaagaaaca tattctgaat ctggccaaag gcaaaggcag
     2581 gatcagcaag accaagaagg agtacctcct cgaagaacgc gacattaaca gatttagtgt
     2641 gcagaaagat ttcatcaacc gaaaccttgt cgatactcgg tacgccacga gaggcctgat
     2701 gaatctcctc aggagctact tccgcgtcaa taatctggac gttaaagtca agagcataaa
     2761 tgggggattc accagctttc tgaggagaaa gtggaagttt aagaaggaac gaaacaaagg
     2821 atacaagcac catgctgagg atgctttgat catcgctaac gcggacttta tctttaagga
     2881 atggaaaaag ctggataagg caaagaaagt gatggaaaac cagatgttcg aggagaagca
     2941 ggcagagtca atgcctgaga tcgagacaga gcaggaatac aaggaaattt tcatcacccc
     3001 tcatcagatt aaacacataa aggacttcaa agactataaa tactctcata gggtggacaa
     3061 aaaacccaat cgcgagctca ttaatgacac cctgtactca acacggaagg atgataaagg
     3121 taataccttg attgtgaata atcttaatgg attgtatgac aaagataacg acaagctcaa
     3181 gaagctgatc aacaagtctc cagagaagct ccttatgtat caccacgacc cacagactta
     3241 tcagaaattg aaactgatca tggagcaata cggggatgag aagaacccac tctacaaata
     3301 ttatgaggaa acaggtaatt acctgaccaa gtactccaag aaggataacg gaccagtgat
     3361 caaaaagata aagtactatg gcaacaaact taatgcgcat ttggacataa ctgacgatta
     3421 ccccaattct cgaaacaagg ttgtgaagct ctccctgaag ccttatagat ttgacgtgta
     3481 cctggataat ggggtttata aattcgtcac cgtgaaaaat ctggacgtga tcaaaaagga
     3541 gaactattat gaagtaaact caaagtgcta tgaggaggcg aagaagctga agaagatctc
     3601 caatcaggcc gagttcatcg cttccttcta taataacgat ctcatcaaga tcaatggaga
     3661 gctttatcgc gtcattggtg tgaacaatga cttgctgaac aggatcgaag tcaatatgat
     3721 agacattacc taccgggagt atctcgaaaa catgaatgat aaacggccgc ctcgcatcat
     3781 caagacaatc gcatctaaaa ctcagtcaat aaaaaagtac tctaccgata tcctggggaa
     3841 tctctatgaa gtgaagtcaa agaagcaccc acaaatcatt aaaaaaggtg gatccgcaga
     3901 agcagcagca aaggaagcag cagcaaagga agcagcagca aaggcagtcg acaccggtcc
     3961 agcagctaag agagttaaac tagattagcg gccgcatcgt caggccaagc ttccatcgat
     4021 agacatgata agatacattg atgagtttgg acaaaccaca acaagaatgc agtgaaaaaa
     4081 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4141 taaacaagtt aacaacaaca attgcattca ttttatgttt caggtaccga gggcctattt
     4201 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
     4261 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     4321 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     4381 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgga
     4441 gaccaaggca gttttttgtt ttagtactct ggaaacagaa tctactaaaa caaggcaaaa
     4501 tgccgtgttt atctcgtcaa cttgttggcg agatttttgc ggccgcagga acccctagtg
     4561 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4621 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4681 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4741 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4801 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4861 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4921 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4981 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5041 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5101 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5161 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     5221 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5281 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5341 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5401 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     5461 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5521 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5581 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5641 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5701 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5761 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5821 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5881 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5941 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6001 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6061 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6121 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6181 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6241 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6301 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     6361 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6421 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6481 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6541 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6601 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6661 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6721 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6781 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6841 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6901 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6961 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     7021 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7081 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7141 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7201 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7261 tttgctggcc ttttgctcac atgt
//
