LOCUS       sequence-250844-        7426 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 7426)
  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 7426)
  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-Chrnb4-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7426
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     mobile_element  191..356
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      540..994
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(627..646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     836..857
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1020..1124
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1125..1193,1194..1985)
                     /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(1343..1362)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2156..2744
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2645..2664
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  3049..3273
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(3340..3873)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4043..4048,4049..4065,4066..4071))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   4411..4551
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4422..4551
                     /label=AAV2 ITR
     promoter        4576..4816
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     4576..4596
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4747..4766
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4842..4917
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4957..5404
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      5723..5732
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5729..6439
                     /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(5874..5892)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6142..6161
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    6446..7034
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6499..6519)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6917..6928)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7048..7067)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7049..7067)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    7093..7227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7130..7149)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7184..7203
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
       61 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
      121 cgcagctgcc tgcaggtctg agacaataac cctgataaat gcttcaataa tgtaagctct
      181 aggagatccg aaccagataa gtgaaatcta gttccaaact attttgtcat ttttaatttt
      241 cgtattagct tacgacgcta cacccagttc ccatctattt tgtcactctt ccctaaataa
      301 tccttaaaaa ctccatttcc acccctccca gttcccaact attttgtccg cccacagcgg
      361 ggcatttttc ttcctgttat gtttttaatc aaacatcctg ccaactccat gtgacaaacc
      421 gtcatcttcg gctacttttt ctctgtcaca gaatgaaaat ttttctgtca tctcttcgtt
      481 attaatgttt gtaattgact gaatatcaac gcttatttgc agcctgaatg gcgaatggga
      541 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
      601 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
      661 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
      721 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
      781 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct taatagtgga
      841 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
      901 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
      961 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     1021 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1081 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1141 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1201 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1261 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     1321 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     1381 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     1441 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     1501 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     1561 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     1621 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     1681 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     1741 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     1801 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     1861 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     1921 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     1981 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     2041 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2101 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2161 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2221 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2281 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     2341 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2401 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2461 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2521 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2581 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2641 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2701 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2761 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2821 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2881 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2941 attttctcct tacgcatctg tgcggtattt cacaccgcat agaccagccg cgtaacctgg
     3001 caaaatcggt tacggttgag taataaatgg atgccctgcg taagcgggtg tgggcggaca
     3061 ataaagtctt aaactgaaca aaatagatct aaactatgac aataaagtct taaactagac
     3121 agaatagttg taaactgaaa tcagtccagt tatgctgtga aaaagcatac tggacttttg
     3181 ttatggctaa agcaaactct tcattttctg aagtgcaaat tgcccgtcgt attaaagagg
     3241 ggcgtggcca agggcatggt aaagactata ttcgcggcgt tgtgacaatt taccgaacaa
     3301 ctccgcggcc gggaagccga tctcggcttg aacgaattgt taggtggcgg tacttgggtc
     3361 gatatcaaag tgcatcactt cttcccgtat gcccaacttt gtatagagag ccactgcggg
     3421 atcgtcaccg taatctgctt gcacgtagat cacataagca ccaagcgcgt tggcctcatg
     3481 cttgaggaga ttgatgagcg cggtggcaat gccctgcctc cggtgctcgc cggagactgc
     3541 gagatcatag atatagatct cactacgcgg ctgctcaaac ttgggcagaa cgtaagccgc
     3601 gagagcgcca acaaccgctt cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg
     3661 gagcaagttc ccgaggtaat cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc
     3721 gaactcacga ccgaaaagat caagagcagc ccgcatggat ttgacttggt cagggccgag
     3781 cctacatgtg cgaatgatgc ccatacttga gccacctaac tttgttttag ggcgactgcc
     3841 ctgctgcgta acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc
     3901 ttgctgcttg gatgcccgag gcatagactg tacaaaaaaa cagtcataac aagccatgaa
     3961 aaccgccact gcgccgttac caccgctgcg ttcggtcaag gttctggacc agttgcgtga
     4021 gcgcatacgc tacttgcatt acagtttacg aaccgaacag gcttatgtca actgggttcg
     4081 tgccttcatc cgtttccacg gtgtgcgtca cccggcaacc ttgggcagca gcgaagtcga
     4141 ggcatttctg tcctggctgg cgaacgagcg caaggtttcg gtctccacgc atcgtcaggc
     4201 attggcggcc ttgctgttct tctacggcaa ggtgctgtgc acggatctgc cctggcttca
     4261 ggagatcgga agacctcggc cgtcgcggcg cttgccggtg gtgctgaccc cggatgaagt
     4321 ggttcgcatc ctcggttttc tggaaggcga gcatcgtttg ttcgcccagg actctagcta
     4381 tagttctagt ggttggctac agcttgcatg cctgcaggca gctgcgcgct cgctcgctca
     4441 ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
     4501 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgca
     4561 cgcgtcccct tcaccgaggg cctatttccc atgattcctt catatttgca tatacgatac
     4621 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     4681 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     4741 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     4801 atcttgtgga aaggacgaaa cggactgact accgcctggc cgttttagag ctagaaatag
     4861 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     4921 tttactagtt ctagactgca gagggccctg cgtatgagtg caagtgggtt ttaggaccag
     4981 gatgaggcgg ggtgggggtg cctacctgac gaccgacccc gacccactgg acaagcaccc
     5041 aacccccatt ccccaaattg cgcatcccct atcagagagg gggaggggaa acaggatgcg
     5101 gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca gtgccttcgc ccccgcctgg
     5161 cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg acgtcactcg ccggtccccc
     5221 gcaaactccc cttcccggcc accttggtcg cgtccgcgcc gccgccggcc cagccggacc
     5281 gcaccacgcg aggcgcgaga taggggggca cgggcgcgac catctgcgct gcggcgccgg
     5341 cgactcagcg ctgcctcagt ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc
     5401 gcagtcgaat tcaagctgct agctagtacc ggttcgaaca ggtaagcgcc cctaaaatcc
     5461 ctttgggcac aatgtgtcct gaggggagag gcagcgacct gtagatggga cgggggcact
     5521 aaccctcagg tttggggctt ctgaatgtga gtatcgccat gtaagcccag tatttggcca
     5581 atctcagaaa gctcctggtc cctggaggga tggagagaga aaaacaaaca gctcctggag
     5641 cagggagagt gctggcctct tgctctccgg ctccctctgt tgccctctgg tttctcccca
     5701 ggttgaattc gatatcggat ccgccaccat ggtgagcaag ggcgaggagg ataacatggc
     5761 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     5821 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     5881 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     5941 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     6001 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     6061 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     6121 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     6181 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     6241 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     6301 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     6361 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     6421 ggacgagctg tacaagtaac tcgagaatca acctctggat tacaaaattt gtgaaagatt
     6481 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     6541 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     6601 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     6661 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     6721 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     6781 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     6841 gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     6901 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     6961 ggctctgcgg cctcttccgc gtcttcgcct ccgccctcag acgagtcgga tctccctttg
     7021 ggccgcctcc ccgcggtacc caattcgccc tatagtgagt cgtattacgc gcgcagcggc
     7081 cgaccatggc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     7141 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     7201 atcaatgtat cttatcatgt ctggatctcc ggacacgtgc ggaccgagcg gccgcaggaa
     7261 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccgcc
     7321 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggcatgca
     7381 agctgtagcc aaccactaga actatagcta gagtcctggg cgaaca
//
