LOCUS       sequence-89642-e        7312 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 promoter-driven single guide RNA complementary to the human AAVS1
            locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7312)
  AUTHORS   Maggio I, Holkers M, Liu J, Janssen JM, Chen X, Goncalves MA
  TITLE     Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease 
            complexes induces targeted mutagenesis in a diverse array of human 
            cells.
  JOURNAL   Sci Rep. 2014 May 29;4:5105. doi: 10.1038/srep05105.
  PUBMED    24870050
REFERENCE   2  (bases 1 to 7312)
  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: "Sci Rep."; 
            date: "2014-05-29"; volume: "4:5105. doi"; pages: " 
            10.1038/srep05105"
COMMENT     Sequence Label: pAdSh.U6.gRNAS1
FEATURES             Location/Qualifiers
     source          1..7312
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..103
                     /label=ITR
                     /note="inverted terminal repeat of human adenovirus 
                     serotype 5"
     misc_signal     191..341
                     /label=Ad5 Psi
                     /note="packaging signal for adenovirus serotype 5"
     promoter        397..645
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     397..417
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     568..587
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        666..741
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(753..777)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    867..988
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(878..897)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     932..951
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4272..4374
                     /label=ITR
                     /note="inverted terminal repeat of human adenovirus 
                     serotype 5"
     primer_bind     complement(4463..4480)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4634..5222)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4714..4733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             6054..6848
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             6054..6848
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6108..6127)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6718..6737
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(6896..7275)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6958..6979)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7170..7189
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt
       61 ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt
      121 gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg
      181 gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag
      241 taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga
      301 agtgaaatct gaataatttt gtgttactca tagcgcgtaa tactggtacg taccgcatgc
      361 gatctagctt gcatgcgtac caaggtcggg caggaagagg gcctatttcc catgattcct
      421 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
      481 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
      541 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
      601 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggggc cactagggac
      661 aggatgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
      721 aaagtggcac cgagtcggtg ctttttttct agacccagct ttcttgtaca aagttggcat
      781 taaagggcga attccagcac actggcggcc gttactacga ccgcggacat gtacagagct
      841 cgagcggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
      901 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
      961 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     1021 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa
     1081 aatcgataag gatctgggcg tggttaaggg tgggaaagaa tatataaggt gggggtctta
     1141 tgtagttttg tatctgtttt gcagcagccg ccgccgccat gagcaccaac tcgtttgatg
     1201 gaagcattgt gagctcatat ttgacaacgc gcatgccccc atgggccggg gtgcgtcaga
     1261 atgtgatggg ctccagcatt gatggtcgcc ccgtcctgcc cgcaaactct actaccttga
     1321 cctacgagac cgtgtctgga acgccgttgg agactgcagc ctccgccgcc gcttcagccg
     1381 ctgcagccac cgcccgcggg attgtgactg actttgcttt cctgagcccg cttgcaagca
     1441 gtgcagcttc ccgttcatcc gcccgcgatg acaagttgac ggctcttttg gcacaattgg
     1501 attctttgac ccgggaactt aatgtcgttt ctcagcagct gttggatctg cgccagcagg
     1561 tttctgccct gaaggcttcc tcccctccca atgcggttta aaacataaat aaaaaaccag
     1621 actctgtttg gatttggatc aagcaagtgt cttgctgtct ttatttaggg gttttgcgcg
     1681 cgcggtaggc ccgggaccag cggtctcggt cgttgagggt cctgtgtatt ttttccagga
     1741 cgtggtaaag gtgactctgg atgttcagat acatgggcat aagcccgtct ctggggtgga
     1801 ggtagcacca ctgcagagct tcatgctgcg gggtggtgtt gtagatgatc cagtcgtagc
     1861 aggagcgctg ggcgtggtgc ctaaaaatgt ctttcagtag caagctgatt gccaggggca
     1921 ggcccttggt gtaagtgttt acaaagcggt taagctggga tgggtgcata cgtggggata
     1981 tgagatgcat cttggactgt atttttaggt tggctatgtt cccagccata tccctccggg
     2041 gattcatgtt gtgcagaacc accagcacag tgtatccggt gcacttggga aatttgtcat
     2101 gtagcttaga aggaaatgcg tggaagaact tggagacgcc cttgtgacct ccaagatttt
     2161 ccatgcattc gtccataatg atggcaatgg gcccacgggc ggcggcctgg gcgaagatat
     2221 ttctgggatc actaacgtca tagttgtgtt ccaggatgag atcgtcatag gccattttta
     2281 caaagcgcgg gcggagggtg ccagactgcg gtataatggt tccatccggc ccaggggcgt
     2341 agttaccctc acagatttgc atttcccacg ctttgagttc agatgggggg atcatgtcta
     2401 cctgcggggc gatgaagaaa acggtttccg gggtagggga gatcagctgg gaagaaagca
     2461 ggttcctgag cagctgcgac ttaccgcagc cggtgggccc gtaaatcaca cctattaccg
     2521 ggtgcaactg gtagttaaga gagctgcagc tgccgtcatc cctgagcagg ggggccactt
     2581 cgttaagcat gtccctgact cgcatgtttt ccctgaccaa atccgccaga aggcgctcgc
     2641 cgcccagcga tagcagttct tgcaaggaag caaagttttt caacggtttg agaccgtccg
     2701 ccgtaggcat gcttttgagc gtttgaccaa gcagttccag gcggtcccac agctcggtca
     2761 cctgctctac ggcatctcga tccagcatat ctcctcgttt cgcgggttgg ggcggctttc
     2821 gctgtacggc agtagtcggt gctcgtccag acgggccagg gtcatgtctt tccacgggcg
     2881 cagggtcctc gtcagcgtag tctgggtcac ggtgaagggg tgcgctccgg gctgcgcgct
     2941 ggccagggtg cgcttgaggc tggtcctgct ggtgctgaag cgctgccggt cttcgccctg
     3001 cgcgtcggcc aggtagcatt tgaccatggt gtcatagtcc agcccctccg cggcgtggcc
     3061 cttggcgcgc agcttgccct tggaggaggc gccgcacgag gggcagtgca gacttttgag
     3121 ggcgtagagc ttgggcgcga gaaataccga ttccggggag taggcatccg cgccgcaggc
     3181 cccgcagacg gtctcgcatt ccacgagcca ggtgagctct ggccgttcgg ggtcaaaaac
     3241 caggtttccc ccatgctttt tgatgcgttt cttacctctg gtttccatga gccggtgtcc
     3301 acgctcggtg acgaaaaggc tgtccgtgtc cccgtataca gacttgagag ggagtttaaa
     3361 cgaattcaat agcttgttgc atgggcggcg atataaaatg caaggtgctg ctcaaaaaat
     3421 caggcaaagc ctcgcgcaaa aaagaaagca catcgtagtc atgctcatgc agataaaggc
     3481 aggtaagctc cggaaccacc acagaaaaag acaccatttt tctctcaaac atgtctgcgg
     3541 gtttctgcat aaacacaaaa taaaataaca aaaaaacatt taaacattag aagcctgtct
     3601 tacaacagga aaaacaaccc ttataagcat aagacggact acggccatgc cggcgtgacc
     3661 gtaaaaaaac tggtcaccgt gattaaaaag caccaccgac agctcctcgg tcatgtccgg
     3721 agtcataatg taagactcgg taaacacatc aggttgattc atcggtcagt gctaaaaagc
     3781 gaccgaaata gcccggggga atacataccc gcaggcgtag agacaacatt acagccccca
     3841 taggaggtat aacaaaatta ataggagaga aaaacacata aacacctgaa aaaccctcct
     3901 gcctaggcaa aatagcaccc tcccgctcca gaacaacata cagcgcttca cagcggcagc
     3961 ctaacagtca gccttaccag taaaaaagaa aacctattaa aaaaacacca ctcgacacgg
     4021 caccagctca atcagtcaca gtgtaaaaaa gggccaagtg cagagcgagt atatatagga
     4081 ctaaaaaatg acgtaacggt taaagtccac aaaaaacacc cagaaaaccg cacgcgaacc
     4141 tacgcccaga aacgaaagcc aaaaaaccca caacttcctc aaatcgtcac ttccgttttc
     4201 ccacgttacg taacttccca ttttaagaaa actacaattc ccaacacata caagttactc
     4261 cgccctaaaa cctacgtcac ccgccccgtt cccacgcccc gcgccacgtc acaaactcca
     4321 ccccctcatt atcatattgg cttcaatcca aaataaggta tattattgat gatgttaatt
     4381 aacatgcatg gatccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     4441 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4501 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4561 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4621 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4681 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4741 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4801 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4861 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4921 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4981 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5041 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     5101 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5161 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5221 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5281 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5341 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5401 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5461 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5521 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5581 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5641 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5701 ccattgctgc agccatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga
     5761 aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga
     5821 caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat
     5881 agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct
     5941 ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct
     6001 gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg
     6061 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     6121 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     6181 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     6241 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     6301 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     6361 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     6421 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     6481 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     6541 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     6601 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     6661 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     6721 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     6781 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     6841 tcttctgaat tttgttaaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     6901 atcggcaaca tcccttataa atcaaaagaa tagaccgcga tagggttgag tgttgttcca
     6961 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     7021 gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttgcggtcg
     7081 aggtgccgta aagctctaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     7141 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     7201 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgcgcg cttaatgcgc
     7261 cgctacaggg cgcgtccatt cgccattcag gatcgaatta attcttaatt aa
//
