LOCUS       sequence-250846-        7450 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 7450)
  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 7450)
  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-Chrna4-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7450
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     mobile_element  168..333
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      517..971
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(604..623)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     813..834
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        997..1101
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1102..1170,1171..1962)
                     /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(1320..1339)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2133..2721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2622..2641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  3026..3250
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(3317..3850)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4020..4025,4026..4042,4043..4048))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   4388..4528
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4399..4528
                     /label=AAV2 ITR
     promoter        4553..4793
                     /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     4553..4573
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4724..4743
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4819..4894
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4934..5381
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      5700..5709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5706..6416
                     /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(5851..5869)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6119..6138
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    6423..7011
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6476..6496)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6894..6905)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7025..7044)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7026..7044)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    7070..7204
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7107..7126)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7161..7180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
       61 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggtctgaga
      121 caataaccct gataaatgct tcaataatgt aagctctagg agatccgaac cagataagtg
      181 aaatctagtt ccaaactatt ttgtcatttt taattttcgt attagcttac gacgctacac
      241 ccagttccca tctattttgt cactcttccc taaataatcc ttaaaaactc catttccacc
      301 cctcccagtt cccaactatt ttgtccgccc acagcggggc atttttcttc ctgttatgtt
      361 tttaatcaaa catcctgcca actccatgtg acaaaccgtc atcttcggct actttttctc
      421 tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt aatgtttgta attgactgaa
      481 tatcaacgct tatttgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa
      541 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      601 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      661 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      721 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      781 gccctttgac gttggagtcc acgttcttaa tagtggactc ttgttccaaa ctggaacaac
      841 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
      901 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
      961 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1021 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     1081 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     1141 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     1201 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     1261 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     1321 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     1381 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     1441 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     1501 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     1561 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     1621 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     1681 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     1741 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     1801 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     1861 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     1921 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     1981 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     2041 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2101 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2161 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2221 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     2281 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2341 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2401 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2461 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2521 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2581 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2641 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2701 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2761 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     2821 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     2881 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     2941 ggtatttcac accgcataga ccagccgcgt aacctggcaa aatcggttac ggttgagtaa
     3001 taaatggatg ccctgcgtaa gcgggtgtgg gcggacaata aagtcttaaa ctgaacaaaa
     3061 tagatctaaa ctatgacaat aaagtcttaa actagacaga atagttgtaa actgaaatca
     3121 gtccagttat gctgtgaaaa agcatactgg acttttgtta tggctaaagc aaactcttca
     3181 ttttctgaag tgcaaattgc ccgtcgtatt aaagaggggc gtggccaagg gcatggtaaa
     3241 gactatattc gcggcgttgt gacaatttac cgaacaactc cgcggccggg aagccgatct
     3301 cggcttgaac gaattgttag gtggcggtac ttgggtcgat atcaaagtgc atcacttctt
     3361 cccgtatgcc caactttgta tagagagcca ctgcgggatc gtcaccgtaa tctgcttgca
     3421 cgtagatcac ataagcacca agcgcgttgg cctcatgctt gaggagattg atgagcgcgg
     3481 tggcaatgcc ctgcctccgg tgctcgccgg agactgcgag atcatagata tagatctcac
     3541 tacgcggctg ctcaaacttg ggcagaacgt aagccgcgag agcgccaaca accgcttctt
     3601 ggtcgaaggc agcaagcgcg atgaatgtct tactacggag caagttcccg aggtaatcgg
     3661 agtccggctg atgttgggag taggtggcta cgtctccgaa ctcacgaccg aaaagatcaa
     3721 gagcagcccg catggatttg acttggtcag ggccgagcct acatgtgcga atgatgccca
     3781 tacttgagcc acctaacttt gttttagggc gactgccctg ctgcgtaaca tcgttgctgc
     3841 tccataacat caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca
     3901 tagactgtac aaaaaaacag tcataacaag ccatgaaaac cgccactgcg ccgttaccac
     3961 cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgagcg catacgctac ttgcattaca
     4021 gtttacgaac cgaacaggct tatgtcaact gggttcgtgc cttcatccgt ttccacggtg
     4081 tgcgtcaccc ggcaaccttg ggcagcagcg aagtcgaggc atttctgtcc tggctggcga
     4141 acgagcgcaa ggtttcggtc tccacgcatc gtcaggcatt ggcggccttg ctgttcttct
     4201 acggcaaggt gctgtgcacg gatctgccct ggcttcagga gatcggaaga cctcggccgt
     4261 cgcggcgctt gccggtggtg ctgaccccgg atgaagtggt tcgcatcctc ggttttctgg
     4321 aaggcgagca tcgtttgttc gcccaggact ctagctatag ttctagtggt tggctacagc
     4381 ttgcatgcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
     4441 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
     4501 gtggccaact ccatcactag gggttcctgc ggccgcacgc gtccccttca ccgagggcct
     4561 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
     4621 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     4681 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     4741 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacga
     4801 tgtggtcctt gtccgcttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     4861 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt actagttcta gactgcagag
     4921 ggccctgcgt atgagtgcaa gtgggtttta ggaccaggat gaggcggggt gggggtgcct
     4981 acctgacgac cgaccccgac ccactggaca agcacccaac ccccattccc caaattgcgc
     5041 atcccctatc agagaggggg aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag
     5101 cttcagcacc gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca
     5161 gcactgaagg cgcgctgacg tcactcgccg gtcccccgca aactcccctt cccggccacc
     5221 ttggtcgcgt ccgcgccgcc gccggcccag ccggaccgca ccacgcgagg cgcgagatag
     5281 gggggcacgg gcgcgaccat ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg
     5341 cggtgggcag cggaggagtc gtgtcgtgcc tgagagcgca gtcgaattca agctgctagc
     5401 tagtaccggt tcgaacaggt aagcgcccct aaaatccctt tgggcacaat gtgtcctgag
     5461 gggagaggca gcgacctgta gatgggacgg gggcactaac cctcaggttt ggggcttctg
     5521 aatgtgagta tcgccatgta agcccagtat ttggccaatc tcagaaagct cctggtccct
     5581 ggagggatgg agagagaaaa acaaacagct cctggagcag ggagagtgct ggcctcttgc
     5641 tctccggctc cctctgttgc cctctggttt ctccccaggt tgaattcgat atcggatccg
     5701 ccaccatggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     5761 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     5821 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     5881 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     5941 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     6001 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     6061 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     6121 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     6181 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     6241 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     6301 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     6361 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaactcg
     6421 agaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     6481 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     6541 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     6601 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     6661 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     6721 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     6781 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     6841 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     6901 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     6961 ttcgcctccg ccctcagacg agtcggatct ccctttgggc cgcctccccg cggtacccaa
     7021 ttcgccctat agtgagtcgt attacgcgcg cagcggccga ccatggccca acttgtttat
     7081 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     7141 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     7201 gatctccgga cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac
     7261 tccctctctg cgcgctcgct cgctcactga ggccgcccgg gctttgcccg ggcggcctca
     7321 gtgagcgagc gagcgcgcag ctgcctgcag gcatgcaagc tgtagccaac cactagaact
     7381 atagctagag tcctgggcga acaaacgatg ctcgccttca ggttttactt gctttaaaaa
     7441 acctcccaca
//
