LOCUS       Exported                7218 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Destination vector for U6::gRNA expression cassette.
ACCESSION   .
VERSION     .
KEYWORDS    AAV-Stuffer
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7218)
  AUTHORS   Thakore PI, Kwon JB, Nelson CE, Rouse DC, Gemberling MP, Oliver ML, 
            Gersbach CA
  TITLE     RNA-guided transcriptional silencing in vivo with S. aureus 
            CRISPR-Cas9 repressors.
  JOURNAL   Nat Commun. 2018 Apr 26;9(1):1674. doi: 10.1038/s41467-018-04048-4.
  PUBMED    29700298
REFERENCE   2  (bases 1 to 7218)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7218
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        182..485
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        486..689
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     640..660
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     686..710
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     polyA_signal    2675..2809
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2699..2718)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2753..2772
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             3159..3962
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3222..3241)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3832..3851
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3994..4011)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4000..4207
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4453..4470)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4624..5212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4704..4723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5383..6243)
                     /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     6006..6025
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6244..6348)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6416..6434
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6472..6494)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6594..6613
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6680..7135)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6817..6838)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7029..7048
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gggggggggg ggggggggtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
       61 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
      121 gcgcgcagag agggagtggc caactccatc actaggggtt cctagatctg aattcggtac
      181 ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      241 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      301 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      361 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      421 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      481 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      541 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      601 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      661 tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac
      721 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccggactc
      781 tagagagacg tacaaaaaag agcaagaagc taaaaaagat ttaaaaatta tttttagcgc
      841 agttaatgga acaggaacta aatttacccc aaaaatatta cgtgaatcag gatataacgt
      901 tattgaggtt gaagagcatg catttgaaga tgaaacattt aaaaatgttg taaatccaaa
      961 tccagaattt gatcctgcat gaaaaatacc gcttgaatat ggtattaaac atgatgcaga
     1021 tattattatt atgaatgacc cagatgctga cagatttgga atggcaataa aacatgatgg
     1081 tcattttgta agattagatg gaaatcaaac aggaccaatt ttaattgatt gaaaattatc
     1141 aaatctaaaa cgcttaaata gcattccaaa aaatccggct ctatattcaa gttttgtaac
     1201 aagtgatttg ggtgatagaa tcgctcatga aaaatatgga gttaatattg taaaaacttt
     1261 aactggattt aaatgaatgg gtagagaaat tgctaaagaa gaagataacg gattaaattt
     1321 tgtttttgct tatgaagaaa gttatggata tgtaattgat gactcagcta gagataaaga
     1381 tggaatacaa gcttctatat taatagcaga ggctgcttga ttttataaaa aacaaaataa
     1441 aacattagta gactatttag aagatttatt taaagaaatg ggtgcatatt acactttcac
     1501 tttaaacttg aattttaaac cagaagaaaa gaaattaaaa attgaaccat taatgaaatc
     1561 attgagagca acacccttaa ctcaaattgc tggacttaaa gttgttaatg ttgaagacta
     1621 catcgatgga atgtataata tgccaggaca agacttacta aaattttatt tagaagataa
     1681 gtcatgattt gctgttcgcc caagtggaac tgaacctaaa ctaaaaattt attttatagg
     1741 tgttggtgaa tctgttcaaa acgctaaagt taaagtagac gaaattatta aagaattaaa
     1801 attaaaaatg aatatatagg agaaaaaatg aaactaaaca aatatataga tcacacatta
     1861 ttaaaacaag atgctacgaa agctgaaatt aaacaattat gtgatgaagc aattgaattt
     1921 gattttgcaa cagtttgtgt taattcatat tgaacaagct attgtaaaga attattaaaa
     1981 ggcacaaatg taggaataac aaatgttgta ggttttcctc taggtgcatg cacaacagct
     2041 acaaaagcat tcgaagtttc tgaagcaatt aaagatggtg caacagaaat tgatatggta
     2101 ttaaatattg gtgcattaaa agacaaaaat tatgaattag ttttagaaga catgaaagct
     2161 gtaaaaaaag cagctggatc acatgttgtt aaatgtatta tggaaaattg tttattaaca
     2221 aaagaagaaa tcatgaaagc ttgtgaaata gctgttgaag ctggattaga atttgttaaa
     2281 acatcaacag gattttcaaa atcaggtgca acatttgaag atgttaaact aatgaagtca
     2341 gttgttaaag acaatgcttt agttaaagca gctggtggag ttagaacatt tgaagatgct
     2401 caaaaaatga ttgaagcagg agctgaccgc ttaggaacaa gtggtggagt agctattatt
     2461 aaaggtgaag aaaacaacgc gagttactaa aactagcgtt tttttatttt gctcattttt
     2521 attaaaagtt tgcaaaaagg aacataaaaa ttctaattat tgatactaaa gttattaaaa
     2581 agaagatttt ggttgatttt ataaaggtca tagaatataa tattttagca tgtgtatttt
     2641 gtgtgctcat ttacaaccgt ctcgcggccg cggggatcca gacatgataa gatacattga
     2701 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     2761 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     2821 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt agtcgacctc
     2881 gagcagtgtg gttttgcaag aggaagcaaa aagcctctcc acccaggcct ggaatgtttc
     2941 cacccaagtc gaaggcagtg tggttttgca agaggaagca aaaagcctct ccacccaggc
     3001 ctggaatgtt tccacccaat gtcgagcaac cccgcccagc gtcttgtcat tggcgaattc
     3061 gaacacgcag atgcagtcgg ggcggcgcgg tcccaggtcc acttcgcata ttaaggtgac
     3121 gcgtgtggcc tcgaacaccg agcgaccctg cagccaatat gggatcggcc attgaacaag
     3181 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3241 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3301 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     3361 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3421 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3481 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3541 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3601 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3661 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     3721 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     3781 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     3841 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     3901 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     3961 gaggggatcc gtcgactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     4021 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     4081 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4141 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     4201 ctggggagag atctaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4261 ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc
     4321 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac cccccccccc ccccccctgc
     4381 agcccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4441 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4501 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4561 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4621 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4681 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4741 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4801 aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4861 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4921 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4981 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5041 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5101 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5161 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5221 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5281 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5341 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5401 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5461 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5521 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5581 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5641 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5701 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5761 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5821 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5881 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5941 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6001 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6061 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6121 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6181 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6241 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6301 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6361 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     6421 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     6481 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     6541 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     6601 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     6661 gagaaaatac cgcatcagga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat
     6721 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     6781 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     6841 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     6901 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     6961 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     7021 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     7081 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcgcgc
     7141 cattcgccat tcaggctacg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     7201 ttacgccagc tggctgca
//
