LOCUS       sequence-250854-        7413 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  pAAV encoding gRNA sequence for loss-of-function indels.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7413)
  AUTHORS   Peng C, Yan Y, Kim VJ, Engle SE, Berry JN, McIntosh JM, Neve RL, 
            Drenan RM
  TITLE     Gene editing vectors for studying nicotinic acetylcholine receptors 
            in cholinergic transmission.
  JOURNAL   Eur J Neurosci. 2018 May 19. doi: 10.1111/ejn.13957.
  PUBMED    29779223
REFERENCE   2  (bases 1 to 7413)
  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: "Eur J 
            Neurosci. 2018 May 19. doi: 10.1111/ejn.13957."
COMMENT     Sequence Label: pFB-U6-beta2 RD-1-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7413
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     mobile_element  172..337
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      521..975
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(608..627)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     817..838
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1001..1105
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1106..1174,1175..1966)
                     /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(1324..1343)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2137..2725
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2626..2645
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  3030..3254
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(3321..3854)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4024..4029,4030..4046,4047..4052))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   4392..4532
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4403..4532
                     /label=AAV2 ITR
     promoter        4557..4805
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4557..4577
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4728..4747
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4823..4898
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4937..5384
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      5703..5712
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5709..6419
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5854..5872)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6122..6141
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    6426..7014
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6479..6499)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6897..6908)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7028..7047)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7029..7047)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    7073..7207
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7110..7129)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7164..7183
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ttgctccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
       61 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggtct
      121 gagacaataa ccctgataaa tgcttcaata atgtaagctc taggagatcc gaaccagata
      181 agtgaaatct agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct
      241 acacccagtt cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc
      301 cacccctccc agttcccaac tattttgtcc gcccacagcg gggcattttt cttcctgtta
      361 tgtttttaat caaacatcct gccaactcca tgtgacaaac cgtcatcttc ggctactttt
      421 tctctgtcac agaatgaaaa tttttctgtc atctcttcgt tattaatgtt tgtaattgac
      481 tgaatatcaa cgcttatttg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
      541 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
      601 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
      661 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
      721 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
      781 tttcgccctt tgacgttgga gtccacgttc ttaatagtgg actcttgttc caaactggaa
      841 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
      901 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
      961 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1021 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1081 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1141 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1201 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1261 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1321 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     1381 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     1441 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1501 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     1561 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     1621 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     1681 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     1741 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     1801 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     1861 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     1921 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     1981 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2041 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2101 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2161 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2221 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2281 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2341 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2401 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2461 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2521 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2581 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2641 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     2701 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     2761 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     2821 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     2881 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     2941 gtgcggtatt tcacaccgca tagaccagcc gcgtaacctg gcaaaatcgg ttacggttga
     3001 gtaataaatg gatgccctgc gtaagcgggt gtgggcggac aataaagtct taaactgaac
     3061 aaaatagatc taaactatga caataaagtc ttaaactaga cagaatagtt gtaaactgaa
     3121 atcagtccag ttatgctgtg aaaaagcata ctggactttt gttatggcta aagcaaactc
     3181 ttcattttct gaagtgcaaa ttgcccgtcg tattaaagag gggcgtggcc aagggcatgg
     3241 taaagactat attcgcggcg ttgtgacaat ttaccgaaca actccgcggc cgggaagccg
     3301 atctcggctt gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact
     3361 tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct
     3421 tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc
     3481 gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata gatatagatc
     3541 tcactacgcg gctgctcaaa cttgggcaga acgtaagccg cgagagcgcc aacaaccgct
     3601 tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa
     3661 tcggagtccg gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga
     3721 tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt gcgaatgatg
     3781 cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt aacatcgttg
     3841 ctgctccata acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga
     3901 ggcatagact gtacaaaaaa acagtcataa caagccatga aaaccgccac tgcgccgtta
     3961 ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg agcgcatacg ctacttgcat
     4021 tacagtttac gaaccgaaca ggcttatgtc aactgggttc gtgccttcat ccgtttccac
     4081 ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg aggcatttct gtcctggctg
     4141 gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg cattggcggc cttgctgttc
     4201 ttctacggca aggtgctgtg cacggatctg ccctggcttc aggagatcgg aagacctcgg
     4261 ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag tggttcgcat cctcggtttt
     4321 ctggaaggcg agcatcgttt gttcgcccag gactctagct atagttctag tggttggcta
     4381 cagcttgcat gcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa
     4441 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
     4501 gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgtcccc ttcaccgagg
     4561 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     4621 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     4681 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     4741 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     4801 acatcagctt gttatagcgg gagttttaga gctagaaata gcaagttaaa ataaggctag
     4861 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttactagt ctagactgca
     4921 gagggccctg cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg
     4981 cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg
     5041 cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc
     5101 cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc
     5161 tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc
     5221 accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga
     5281 taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt
     5341 ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaat tcaagctgct
     5401 agctagtacc ggttcgaaca ggtaagcgcc cctaaaatcc ctttgggcac aatgtgtcct
     5461 gaggggagag gcagcgacct gtagatggga cgggggcact aaccctcagg tttggggctt
     5521 ctgaatgtga gtatcgccat gtaagcccag tatttggcca atctcagaaa gctcctggtc
     5581 cctggaggga tggagagaga aaaacaaaca gctcctggag cagggagagt gctggcctct
     5641 tgctctccgg ctccctctgt tgccctctgg tttctcccca ggttgaattc gatatcggat
     5701 ccgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     5761 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     5821 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     5881 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     5941 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     6001 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     6061 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     6121 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     6181 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     6241 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     6301 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     6361 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaac
     6421 tcgagaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     6481 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     6541 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     6601 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     6661 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     6721 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     6781 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc
     6841 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     6901 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     6961 gtcttcgcct ccgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcggtacc
     7021 caattcgccc tatagtgagt cgtattacgc gcgcagcggc cgaccatggc ccaacttgtt
     7081 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     7141 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     7201 ctggatctcc ggacacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc
     7261 cactccctct ctgcgcgctc gctcgctcac tgaggccgcc cgggctttgc ccgggcggcc
     7321 tcagtgagcg agcgagcgcg cagctgcctg caggcatgca agctgtagcc aaccactaga
     7381 actatagcta gagtcctggg cgaacaaacg atg
//
