LOCUS       sequence-250830-        7466 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 7466)
  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 7466)
  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-Chrnb3-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7466
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     mobile_element  146..311
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      495..949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(582..601)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     791..812
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        975..1079
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1080..1148,1149..1940)
                     /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(1298..1317)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2111..2699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2600..2619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  3004..3228
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(3295..3828)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3998..4003,4004..4020,4021..4026))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   4391..4531
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4402..4531
                     /label=AAV2 ITR
     promoter        4556..4804
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4556..4576
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4727..4746
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4822..4897
                     /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 tataatgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
       61 gtgagcgagc gagcgcgcag ctgcctgcag gtctgagaca ataaccctga taaatgcttc
      121 aataatgtaa gctctaggag atccgaacca gataagtgaa atctagttcc aaactatttt
      181 gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca
      241 ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt
      301 gtccgcccac agcggggcat ttttcttcct gttatgtttt taatcaaaca tcctgccaac
      361 tccatgtgac aaaccgtcat cttcggctac tttttctctg tcacagaatg aaaatttttc
      421 tgtcatctct tcgttattaa tgtttgtaat tgactgaata tcaacgctta tttgcagcct
      481 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
      541 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
      601 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
      661 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
      721 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
      781 gttcttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
      841 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
      901 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
      961 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1021 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1081 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1141 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1201 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1261 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1321 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1381 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1441 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1501 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1561 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1621 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1681 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     1741 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     1801 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     1861 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     1921 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     1981 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2041 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2101 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2161 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2221 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2281 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2341 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2401 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2461 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2521 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2581 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2641 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2701 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2761 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2821 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2881 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatagacc
     2941 agccgcgtaa cctggcaaaa tcggttacgg ttgagtaata aatggatgcc ctgcgtaagc
     3001 gggtgtgggc ggacaataaa gtcttaaact gaacaaaata gatctaaact atgacaataa
     3061 agtcttaaac tagacagaat agttgtaaac tgaaatcagt ccagttatgc tgtgaaaaag
     3121 catactggac ttttgttatg gctaaagcaa actcttcatt ttctgaagtg caaattgccc
     3181 gtcgtattaa agaggggcgt ggccaagggc atggtaaaga ctatattcgc ggcgttgtga
     3241 caatttaccg aacaactccg cggccgggaa gccgatctcg gcttgaacga attgttaggt
     3301 ggcggtactt gggtcgatat caaagtgcat cacttcttcc cgtatgccca actttgtata
     3361 gagagccact gcgggatcgt caccgtaatc tgcttgcacg tagatcacat aagcaccaag
     3421 cgcgttggcc tcatgcttga ggagattgat gagcgcggtg gcaatgccct gcctccggtg
     3481 ctcgccggag actgcgagat catagatata gatctcacta cgcggctgct caaacttggg
     3541 cagaacgtaa gccgcgagag cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat
     3601 gaatgtctta ctacggagca agttcccgag gtaatcggag tccggctgat gttgggagta
     3661 ggtggctacg tctccgaact cacgaccgaa aagatcaaga gcagcccgca tggatttgac
     3721 ttggtcaggg ccgagcctac atgtgcgaat gatgcccata cttgagccac ctaactttgt
     3781 tttagggcga ctgccctgct gcgtaacatc gttgctgctc cataacatca aacatcgacc
     3841 cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata gactgtacaa aaaaacagtc
     3901 ataacaagcc atgaaaaccg ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct
     3961 ggaccagttg cgtgagcgca tacgctactt gcattacagt ttacgaaccg aacaggctta
     4021 tgtcaactgg gttcgtgcct tcatccgttt ccacggtgtg cgtcacccgg caaccttggg
     4081 cagcagcgaa gtcgaggcat ttctgtcctg gctggcgaac gagcgcaagg tttcggtctc
     4141 cacgcatcgt caggcattgg cggccttgct gttcttctac ggcaaggtgc tgtgcacgga
     4201 tctgccctgg cttcaggaga tcggaagacc tcggccgtcg cggcgcttgc cggtggtgct
     4261 gaccccggat gaagtggttc gcatcctcgg ttttctggaa ggcgagcatc gtttgttcgc
     4321 ccaggactct agctatagtt ctagtggttg gctacagctt gcatgcntnn atcagggtta
     4381 ttgtctcaga cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag
     4441 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag
     4501 ggagtggcca actccatcac taggggttcc tgcggccgca cgcgtcccct tcaccgaggg
     4561 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     4621 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     4681 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     4741 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     4801 ccactgcggg actcagctca ggttttagag ctagaaatag caagttaaaa taaggctagt
     4861 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttactagtt 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 atgctcgcct tccagaaaac cgaggatgcg
     7441 aaccacttca tccggggtca gcacca
//
