LOCUS       sequence-89656-e        7423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 promoter-driven single guide RNA complementary to eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7423)
  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 7423)
  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.gRNAGFP
FEATURES             Location/Qualifiers
     source          1..7423
                     /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        532..780
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     532..552
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     703..722
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        801..876
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(888..912)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    978..1099
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(989..1008)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1043..1062
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4383..4485
                     /label=ITR
                     /note="inverted terminal repeat of human adenovirus 
                     serotype 5"
     primer_bind     complement(4574..4591)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4745..5333)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4825..4844)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             6165..6959
                     /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             6165..6959
                     /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(6219..6238)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6829..6848
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(7007..7386)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7069..7090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7281..7300
                     /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 tactggtacc ggttgttaac
      361 gttagccggc tacgtatact ccggaatatt aataggccta ggatgcatat ggcggccgcc
      421 tgcagctggc gccatcgata cgcgtacgtc gaattcgccc tttgtacaaa aaagcaggct
      481 ttaaaggaac caattcagtc gactggatcc ggtaccaagg tcgggcagga agagggccta
      541 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga
      601 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
      661 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
      721 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
      781 ggagcgcacc atcttcttca gttttagagc tagaaatagc aagttaaaat aaggctagtc
      841 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctagacc cagctttctt
      901 gtacaaagtt ggcattaaag ggcgaattcg accgcggaca tgtacagagc tcgagcggcc
      961 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     1021 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     1081 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     1141 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa
     1201 ggatctgggc gtggttaagg gtgggaaaga atatataagg tgggggtctt atgtagtttt
     1261 gtatctgttt tgcagcagcc gccgccgcca tgagcaccaa ctcgtttgat ggaagcattg
     1321 tgagctcata tttgacaacg cgcatgcccc catgggccgg ggtgcgtcag aatgtgatgg
     1381 gctccagcat tgatggtcgc cccgtcctgc ccgcaaactc tactaccttg acctacgaga
     1441 ccgtgtctgg aacgccgttg gagactgcag cctccgccgc cgcttcagcc gctgcagcca
     1501 ccgcccgcgg gattgtgact gactttgctt tcctgagccc gcttgcaagc agtgcagctt
     1561 cccgttcatc cgcccgcgat gacaagttga cggctctttt ggcacaattg gattctttga
     1621 cccgggaact taatgtcgtt tctcagcagc tgttggatct gcgccagcag gtttctgccc
     1681 tgaaggcttc ctcccctccc aatgcggttt aaaacataaa taaaaaacca gactctgttt
     1741 ggatttggat caagcaagtg tcttgctgtc tttatttagg ggttttgcgc gcgcggtagg
     1801 cccgggacca gcggtctcgg tcgttgaggg tcctgtgtat tttttccagg acgtggtaaa
     1861 ggtgactctg gatgttcaga tacatgggca taagcccgtc tctggggtgg aggtagcacc
     1921 actgcagagc ttcatgctgc ggggtggtgt tgtagatgat ccagtcgtag caggagcgct
     1981 gggcgtggtg cctaaaaatg tctttcagta gcaagctgat tgccaggggc aggcccttgg
     2041 tgtaagtgtt tacaaagcgg ttaagctggg atgggtgcat acgtggggat atgagatgca
     2101 tcttggactg tatttttagg ttggctatgt tcccagccat atccctccgg ggattcatgt
     2161 tgtgcagaac caccagcaca gtgtatccgg tgcacttggg aaatttgtca tgtagcttag
     2221 aaggaaatgc gtggaagaac ttggagacgc ccttgtgacc tccaagattt tccatgcatt
     2281 cgtccataat gatggcaatg ggcccacggg cggcggcctg ggcgaagata tttctgggat
     2341 cactaacgtc atagttgtgt tccaggatga gatcgtcata ggccattttt acaaagcgcg
     2401 ggcggagggt gccagactgc ggtataatgg ttccatccgg cccaggggcg tagttaccct
     2461 cacagatttg catttcccac gctttgagtt cagatggggg gatcatgtct acctgcgggg
     2521 cgatgaagaa aacggtttcc ggggtagggg agatcagctg ggaagaaagc aggttcctga
     2581 gcagctgcga cttaccgcag ccggtgggcc cgtaaatcac acctattacc gggtgcaact
     2641 ggtagttaag agagctgcag ctgccgtcat ccctgagcag gggggccact tcgttaagca
     2701 tgtccctgac tcgcatgttt tccctgacca aatccgccag aaggcgctcg ccgcccagcg
     2761 atagcagttc ttgcaaggaa gcaaagtttt tcaacggttt gagaccgtcc gccgtaggca
     2821 tgcttttgag cgtttgacca agcagttcca ggcggtccca cagctcggtc acctgctcta
     2881 cggcatctcg atccagcata tctcctcgtt tcgcgggttg gggcggcttt cgctgtacgg
     2941 cagtagtcgg tgctcgtcca gacgggccag ggtcatgtct ttccacgggc gcagggtcct
     3001 cgtcagcgta gtctgggtca cggtgaaggg gtgcgctccg ggctgcgcgc tggccagggt
     3061 gcgcttgagg ctggtcctgc tggtgctgaa gcgctgccgg tcttcgccct gcgcgtcggc
     3121 caggtagcat ttgaccatgg tgtcatagtc cagcccctcc gcggcgtggc ccttggcgcg
     3181 cagcttgccc ttggaggagg cgccgcacga ggggcagtgc agacttttga gggcgtagag
     3241 cttgggcgcg agaaataccg attccgggga gtaggcatcc gcgccgcagg ccccgcagac
     3301 ggtctcgcat tccacgagcc aggtgagctc tggccgttcg gggtcaaaaa ccaggtttcc
     3361 cccatgcttt ttgatgcgtt tcttacctct ggtttccatg agccggtgtc cacgctcggt
     3421 gacgaaaagg ctgtccgtgt ccccgtatac agacttgaga gggagtttaa acgaattcaa
     3481 tagcttgttg catgggcggc gatataaaat gcaaggtgct gctcaaaaaa tcaggcaaag
     3541 cctcgcgcaa aaaagaaagc acatcgtagt catgctcatg cagataaagg caggtaagct
     3601 ccggaaccac cacagaaaaa gacaccattt ttctctcaaa catgtctgcg ggtttctgca
     3661 taaacacaaa ataaaataac aaaaaaacat ttaaacatta gaagcctgtc ttacaacagg
     3721 aaaaacaacc cttataagca taagacggac tacggccatg ccggcgtgac cgtaaaaaaa
     3781 ctggtcaccg tgattaaaaa gcaccaccga cagctcctcg gtcatgtccg gagtcataat
     3841 gtaagactcg gtaaacacat caggttgatt catcggtcag tgctaaaaag cgaccgaaat
     3901 agcccggggg aatacatacc cgcaggcgta gagacaacat tacagccccc ataggaggta
     3961 taacaaaatt aataggagag aaaaacacat aaacacctga aaaaccctcc tgcctaggca
     4021 aaatagcacc ctcccgctcc agaacaacat acagcgcttc acagcggcag cctaacagtc
     4081 agccttacca gtaaaaaaga aaacctatta aaaaaacacc actcgacacg gcaccagctc
     4141 aatcagtcac agtgtaaaaa agggccaagt gcagagcgag tatatatagg actaaaaaat
     4201 gacgtaacgg ttaaagtcca caaaaaacac ccagaaaacc gcacgcgaac ctacgcccag
     4261 aaacgaaagc caaaaaaccc acaacttcct caaatcgtca cttccgtttt cccacgttac
     4321 gtaacttccc attttaagaa aactacaatt cccaacacat acaagttact ccgccctaaa
     4381 acctacgtca cccgccccgt tcccacgccc cgcgccacgt cacaaactcc accccctcat
     4441 tatcatattg gcttcaatcc aaaataaggt atattattga tgatgttaat taacatgcat
     4501 ggatccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     4561 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4621 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4681 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4741 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4801 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4861 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4921 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4981 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5041 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5101 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5161 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     5221 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5281 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5341 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5401 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5461 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5521 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5581 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5641 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5701 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5761 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     5821 cagccatgag attatcaaaa aggatcttca cctagatcct tttcacgtag aaagccagtc
     5881 cgcagaaacg gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa
     5941 acgcaagcgc aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact
     6001 gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg
     6061 ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca
     6121 ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     6181 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     6241 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     6301 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     6361 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     6421 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     6481 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     6541 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     6601 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     6661 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     6721 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     6781 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     6841 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     6901 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa
     6961 ttttgttaaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaac
     7021 atcccttata aatcaaaaga atagaccgcg atagggttga gtgttgttcc agtttggaac
     7081 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     7141 ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttgcggtc gaggtgccgt
     7201 aaagctctaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     7261 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     7321 agtgtagcgg tcacgctgcg cgtaaccacc acacccgcgc gcttaatgcg ccgctacagg
     7381 gcgcgtccat tcgccattca ggatcgaatt aattcttaat taa
//
