LOCUS       sequence-260654-        7192 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Destination vector for U6::gRNA expression cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7192)
  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 7192)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-04-26"; volume: "9(1):1674. doi"; pages: " 
            10.1038/s41467-018-04048-4"
COMMENT     Sequence Label: AAV-Stuffer
FEATURES             Location/Qualifiers
     source          1..7192
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        183..468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        469..672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     623..643
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     669..693
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     polyA_signal    2658..2792
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2682..2701)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2736..2755
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             3141..3944
                     /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(3204..3223)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3814..3833
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3976..3993)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3982..4189
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(4605..5193)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4685..4704)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5364..6155,6156..6224))
                     /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     5987..6006
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6225..6329)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6397..6415
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6453..6475)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6575..6594
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6661..7116)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6798..6819)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7010..7029
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gggggggggg gggggggscc csccmcyccc tctcygcgcs cycgctcgct cactgaggcc
       61 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
      121 gcgcgcagag agggagtggc caactccatc actaggggtt cctcagatct gaattcggta
      181 cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      241 ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      361 gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac
      421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      661 ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
      721 gacctccata gaagacaccg ggaccgatcc agcctccgga ctctagagag acgtacaaaa
      781 aagagcaaga agctaaaaaa gatttaaaaa ttatttttag cgcagttaat ggaacaggaa
      841 ctaaatttac cccaaaaata ttacgtgaat caggatataa cgttattgag gttgaagagc
      901 atgcatttga agatgaaaca tttaaaaatg ttgtaaatcc aaatccagaa tttgatcctg
      961 catgaaaaat accgcttgaa tatggtatta aacatgatgc agatattatt attatgaatg
     1021 acccagatgc tgacagattt ggaatggcaa taaaacatga tggtcatttt gtaagattag
     1081 atggaaatca aacaggacca attttaattg attgaaaatt atcaaatcta aaacgcttaa
     1141 atagcattcc aaaaaatccg gctctatatt caagttttgt aacaagtgat ttgggtgata
     1201 gaatcgctca tgaaaaatat ggagttaata ttgtaaaaac tttaactgga tttaaatgaa
     1261 tgggtagaga aattgctaaa gaagaagata acggattaaa ttttgttttt gcttatgaag
     1321 aaagttatgg atatgtaatt gatgactcag ctagagataa agatggaata caagcttcta
     1381 tattaatagc agaggctgct tgattttata aaaaacaaaa taaaacatta gtagactatt
     1441 tagaagattt atttaaagaa atgggtgcat attacacttt cactttaaac ttgaatttta
     1501 aaccagaaga aaagaaatta aaaattgaac cattaatgaa atcattgaga gcaacaccct
     1561 taactcaaat tgctggactt aaagttgtta atgttgaaga ctacatcgat ggaatgtata
     1621 atatgccagg acaagactta ctaaaatttt atttagaaga taagtcatga tttgctgttc
     1681 gcccaagtgg aactgaacct aaactaaaaa tttattttat aggtgttggt gaatctgttc
     1741 aaaacgctaa agttaaagta gacgaaatta ttaaagaatt aaaattaaaa atgaatatat
     1801 aggagaaaaa atgaaactaa acaaatatat agatcacaca ttattaaaac aagatgctac
     1861 gaaagctgaa attaaacaat tatgtgatga agcaattgaa tttgattttg caacagtttg
     1921 tgttaattca tattgaacaa gctattgtaa agaattatta aaaggcacaa atgtaggaat
     1981 aacaaatgtt gtaggttttc ctctaggtgc atgcacaaca gctacaaaag cattcgaagt
     2041 ttctgaagca attaaagatg gtgcaacaga aattgatatg gtattaaata ttggtgcatt
     2101 aaaagacaaa aattatgaat tagttttaga agacatgaaa gctgtaaaaa aagcagctgg
     2161 atcacatgtt gttaaatgta ttatggaaaa ttgtttatta acaaaagaag aaatcatgaa
     2221 agcttgtgaa atagctgttg aagctggatt agaatttgtt aaaacatcaa caggattttc
     2281 aaaatcaggt gcaacatttg aagatgttaa actaatgaag tcagttgtta aagacaatgc
     2341 tttagttaaa gcagctggtg gagttagaac atttgaagat gctcaaaaaa tgattgaagc
     2401 aggagctgac cgcttaggaa caagtggtgg agtagctatt attaaaggtg aagaaaacaa
     2461 cgcgagttac taaaactagc gtttttttat tttgctcatt tttattaaaa gtttgcaaaa
     2521 aggaacataa aaattctaat tattgatact aaagttatta aaaagaagat tttggttgat
     2581 tttataaagg tcatagaata taatatttta gcatgtgtat tttgtgtgct catttacaac
     2641 cgtctcgcgg ccgcgcggat ccagacatga taagatacat tgatgagttt ggacaaacca
     2701 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     2761 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     2821 ttcaggttca gggggaggtg tgggaggttt tttagtcgac ctcgagcagt gtggttttca
     2881 agaggaagca aaaagcctct ccacccaggc ctggaatgtt tccacccaat gtcgagcagt
     2941 gtggttttgc aagaggaagc aaaaagcctc tccacccagg cctggaatgt ttccacccaa
     3001 tgtcgagcaa accccgccca gcgtcttgtc attggcgaat tcgaacacgc agatgcagtc
     3061 ggggcggcgc ggtcccaggt ccacttcgca tattaaggtg acgcgtgtgg cctcgaacac
     3121 cgagcgaccc tgcagccaat atgggatcgg ccattgaaca agatggattg cacgcaggtt
     3181 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3241 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3301 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3361 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3421 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3481 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3541 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3601 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3661 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3721 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3781 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3841 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3901 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaggggat ccgtcgacta
     3961 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4021 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4081 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4141 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggag agatctgagg
     4201 aacccctagt gatggagttg gccactcccc ccccctgcag cctgcattmw gggggggggg
     4261 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggagaga
     4321 tctgaggaac ccctagtgat ggagttggcc actccccccc cctgcagcct gcattaatga
     4381 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4441 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4501 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4561 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4621 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4681 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4741 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4801 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4861 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4921 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4981 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5041 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5101 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5161 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5221 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5281 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5341 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5401 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5461 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5521 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5581 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5641 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5701 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5761 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5821 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5881 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5941 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6001 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6061 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6121 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6181 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6241 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6301 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6361 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     6421 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     6481 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     6541 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     6601 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     6661 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     6721 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     6781 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     6841 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     6901 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     6961 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     7021 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     7081 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa
     7141 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccaggctg ca
//
