LOCUS       sequence-250852-        7392 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 7392)
  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 7392)
  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-Chrna2-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7392
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     mobile_element  152..317
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      501..955
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(588..607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     797..818
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        981..1085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1086..1154,1155..1946)
                     /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(1304..1323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2117..2705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2606..2625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  3010..3234
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(3301..3834)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4004..4009,4010..4026,4027..4032))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   4372..4512
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4383..4512
                     /label=AAV2 ITR
     promoter        4537..4777
                     /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     4537..4557
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4708..4727
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4803..4878
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4918..5365
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      5684..5693
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5690..6400
                     /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(5835..5853)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6103..6122
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    6407..6995
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6460..6480)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6878..6889)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7009..7028)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7010..7028)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    7054..7188
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7091..7110)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7145..7164
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
       61 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggtct gagacaataa ccctgataaa
      121 tgcttcaata atgtaagctc taggagatcc gaaccagata agtgaaatct agttccaaac
      181 tattttgtca tttttaattt tcgtattagc ttacgacgct acacccagtt cccatctatt
      241 ttgtcactct tccctaaata atccttaaaa actccatttc cacccctccc agttcccaac
      301 tattttgtcc gcccacagcg gggcattttt cttcctgtta tgtttttaat caaacatcct
      361 gccaactcca tgtgacaaac cgtcatcttc ggctactttt tctctgtcac agaatgaaaa
      421 tttttctgtc atctcttcgt tattaatgtt tgtaattgac tgaatatcaa cgcttatttg
      481 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
      541 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
      601 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
      661 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
      721 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
      781 gtccacgttc ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
      841 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
      901 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
      961 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1021 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1081 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1141 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1201 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1261 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     1321 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     1381 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     1441 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     1501 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     1561 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     1621 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     1681 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     1741 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     1801 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     1861 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     1921 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1981 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2041 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2101 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2161 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2221 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     2281 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2341 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2401 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2461 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2521 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2581 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2641 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2701 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2761 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2821 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2881 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2941 tagaccagcc gcgtaacctg gcaaaatcgg ttacggttga gtaataaatg gatgccctgc
     3001 gtaagcgggt gtgggcggac aataaagtct taaactgaac aaaatagatc taaactatga
     3061 caataaagtc ttaaactaga cagaatagtt gtaaactgaa atcagtccag ttatgctgtg
     3121 aaaaagcata ctggactttt gttatggcta aagcaaactc ttcattttct gaagtgcaaa
     3181 ttgcccgtcg tattaaagag gggcgtggcc aagggcatgg taaagactat attcgcggcg
     3241 ttgtgacaat ttaccgaaca actccgcggc cgggaagccg atctcggctt gaacgaattg
     3301 ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact tcttcccgta tgcccaactt
     3361 tgtatagaga gccactgcgg gatcgtcacc gtaatctgct tgcacgtaga tcacataagc
     3421 accaagcgcg ttggcctcat gcttgaggag attgatgagc gcggtggcaa tgccctgcct
     3481 ccggtgctcg ccggagactg cgagatcata gatatagatc tcactacgcg gctgctcaaa
     3541 cttgggcaga acgtaagccg cgagagcgcc aacaaccgct tcttggtcga aggcagcaag
     3601 cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa tcggagtccg gctgatgttg
     3661 ggagtaggtg gctacgtctc cgaactcacg accgaaaaga tcaagagcag cccgcatgga
     3721 tttgacttgg tcagggccga gcctacatgt gcgaatgatg cccatacttg agccacctaa
     3781 ctttgtttta gggcgactgc cctgctgcgt aacatcgttg ctgctccata acatcaaaca
     3841 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa
     3901 acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa
     3961 ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca
     4021 ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac
     4081 cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc
     4141 ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg
     4201 cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt
     4261 ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt
     4321 gttcgcccag gactctagct atagttctag tggttggcta cagcttgcat gcctgcaggc
     4381 agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct
     4441 ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca
     4501 ctaggggttc ctgcggccgc acgcgtcccc ttcaccgagg gcctatttcc catgattcct
     4561 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     4621 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     4681 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     4741 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acgaacaggc ggtcctcggc
     4801 atgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     4861 gtggcaccga gtcggtgctt ttttactagt tctagactgc agagggccct gcgtatgagt
     4921 gcaagtgggt tttaggacca ggatgaggcg gggtgggggt gcctacctga cgaccgaccc
     4981 cgacccactg gacaagcacc caacccccat tccccaaatt gcgcatcccc tatcagagag
     5041 ggggagggga aacaggatgc ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac
     5101 agtgccttcg cccccgcctg gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct
     5161 gacgtcactc gccggtcccc cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc
     5221 cgccgccggc ccagccggac cgcaccacgc gaggcgcgag ataggggggc acgggcgcga
     5281 ccatctgcgc tgcggcgccg gcgactcagc gctgcctcag tctgcggtgg gcagcggagg
     5341 agtcgtgtcg tgcctgagag cgcagtcgaa ttcaagctgc tagctagtac cggttcgaac
     5401 aggtaagcgc ccctaaaatc cctttgggca caatgtgtcc tgaggggaga ggcagcgacc
     5461 tgtagatggg acgggggcac taaccctcag gtttggggct tctgaatgtg agtatcgcca
     5521 tgtaagccca gtatttggcc aatctcagaa agctcctggt ccctggaggg atggagagag
     5581 aaaaacaaac agctcctgga gcagggagag tgctggcctc ttgctctccg gctccctctg
     5641 ttgccctctg gtttctcccc aggttgaatt cgatatcgga tccgccacca tggtgagcaa
     5701 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
     5761 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     5821 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
     5881 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     5941 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     6001 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     6061 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     6121 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     6181 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     6241 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     6301 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     6361 ccgccactcc accggcggca tggacgagct gtacaagtaa ctcgagaatc aacctctgga
     6421 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     6481 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     6541 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     6601 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     6661 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     6721 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     6781 ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac
     6841 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     6901 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc tccgccctca
     6961 gacgagtcgg atctcccttt gggccgcctc cccgcggtac ccaattcgcc ctatagtgag
     7021 tcgtattacg cgcgcagcgg ccgaccatgg cccaacttgt ttattgcagc ttataatggt
     7081 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     7141 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctc cggacacgtg
     7201 cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     7261 cgctcgctca ctgaggccgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     7321 gcagctgcct gcaggcatgc aagctgtagc caaccactag aactatagct agagtcctgg
     7381 gcgaacaaac ga
//
