LOCUS       sequence-250829-        7463 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 7463)
  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 7463)
  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-Chrna6-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7463
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     mobile_element  170..335
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      519..973
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(606..625)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     815..836
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        999..1103
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1104..1172,1173..1964)
                     /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(1322..1341)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2135..2723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2624..2643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  3028..3252
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(3319..3852)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4022..4027,4028..4044,4045..4050))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   4390..4530
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4401..4530
                     /label=AAV2 ITR
     promoter        4555..4795
                     /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     4555..4575
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4726..4745
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4821..4896
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4936..5383
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      5702..5711
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5708..6418
                     /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(5853..5871)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6121..6140
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    6425..7013
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6478..6498)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6896..6907)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7027..7046)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7028..7046)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    7072..7206
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7109..7128)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7163..7182
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
       61 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggtctga
      121 gacaataacc ctgataaatg cttcaataat gtaagctcta ggagatccga accagataag
      181 tgaaatctag ttccaaacta ttttgtcatt tttaattttc gtattagctt acgacgctac
      241 acccagttcc catctatttt gtcactcttc cctaaataat ccttaaaaac tccatttcca
      301 cccctcccag ttcccaacta ttttgtccgc ccacagcggg gcatttttct tcctgttatg
      361 tttttaatca aacatcctgc caactccatg tgacaaaccg tcatcttcgg ctactttttc
      421 tctgtcacag aatgaaaatt tttctgtcat ctcttcgtta ttaatgtttg taattgactg
      481 aatatcaacg cttatttgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
      541 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
      601 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
      661 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
      721 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
      781 tcgccctttg acgttggagt ccacgttctt aatagtggac tcttgttcca aactggaaca
      841 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
      901 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
      961 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1021 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     1081 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     1141 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     1201 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     1261 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     1321 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     1381 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     1441 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     1501 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     1561 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     1621 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     1681 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     1741 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     1801 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     1861 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     1921 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     1981 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     2041 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2101 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2161 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2221 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2281 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2341 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2401 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2461 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2521 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2581 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2641 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2701 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     2761 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     2821 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     2881 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     2941 gcggtatttc acaccgcata gaccagccgc gtaacctggc aaaatcggtt acggttgagt
     3001 aataaatgga tgccctgcgt aagcgggtgt gggcggacaa taaagtctta aactgaacaa
     3061 aatagatcta aactatgaca ataaagtctt aaactagaca gaatagttgt aaactgaaat
     3121 cagtccagtt atgctgtgaa aaagcatact ggacttttgt tatggctaaa gcaaactctt
     3181 cattttctga agtgcaaatt gcccgtcgta ttaaagaggg gcgtggccaa gggcatggta
     3241 aagactatat tcgcggcgtt gtgacaattt accgaacaac tccgcggccg ggaagccgat
     3301 ctcggcttga acgaattgtt aggtggcggt acttgggtcg atatcaaagt gcatcacttc
     3361 ttcccgtatg cccaactttg tatagagagc cactgcggga tcgtcaccgt aatctgcttg
     3421 cacgtagatc acataagcac caagcgcgtt ggcctcatgc ttgaggagat tgatgagcgc
     3481 ggtggcaatg ccctgcctcc ggtgctcgcc ggagactgcg agatcataga tatagatctc
     3541 actacgcggc tgctcaaact tgggcagaac gtaagccgcg agagcgccaa caaccgcttc
     3601 ttggtcgaag gcagcaagcg cgatgaatgt cttactacgg agcaagttcc cgaggtaatc
     3661 ggagtccggc tgatgttggg agtaggtggc tacgtctccg aactcacgac cgaaaagatc
     3721 aagagcagcc cgcatggatt tgacttggtc agggccgagc ctacatgtgc gaatgatgcc
     3781 catacttgag ccacctaact ttgttttagg gcgactgccc tgctgcgtaa catcgttgct
     3841 gctccataac atcaaacatc gacccacggc gtaacgcgct tgctgcttgg atgcccgagg
     3901 catagactgt acaaaaaaac agtcataaca agccatgaaa accgccactg cgccgttacc
     3961 accgctgcgt tcggtcaagg ttctggacca gttgcgtgag cgcatacgct acttgcatta
     4021 cagtttacga accgaacagg cttatgtcaa ctgggttcgt gccttcatcc gtttccacgg
     4081 tgtgcgtcac ccggcaacct tgggcagcag cgaagtcgag gcatttctgt cctggctggc
     4141 gaacgagcgc aaggtttcgg tctccacgca tcgtcaggca ttggcggcct tgctgttctt
     4201 ctacggcaag gtgctgtgca cggatctgcc ctggcttcag gagatcggaa gacctcggcc
     4261 gtcgcggcgc ttgccggtgg tgctgacccc ggatgaagtg gttcgcatcc tcggttttct
     4321 ggaaggcgag catcgtttgt tcgcccagga ctctagctat agttctagtg gttggctaca
     4381 gcttgcatgc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc
     4441 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
     4501 gagtggccaa ctccatcact aggggttcct gcggccgcac gcgtcccctt caccgagggc
     4561 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     4621 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     4681 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     4741 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     4801 gaatgtctcc gatcccgtca gttttagagc tagaaatagc aagttaaaat aaggctagtc
     4861 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttactagttc tagactgcag
     4921 agggccctgc gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc
     4981 ctacctgacg accgaccccg acccactgga caagcaccca acccccattc cccaaattgc
     5041 gcatccccta tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc
     5101 agcttcagca ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct
     5161 cagcactgaa ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca
     5221 ccttggtcgc gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat
     5281 aggggggcac gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc
     5341 tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg cagtcgaatt caagctgcta
     5401 gctagtaccg gttcgaacag gtaagcgccc ctaaaatccc tttgggcaca atgtgtcctg
     5461 aggggagagg cagcgacctg tagatgggac gggggcacta accctcaggt ttggggcttc
     5521 tgaatgtgag tatcgccatg taagcccagt atttggccaa tctcagaaag ctcctggtcc
     5581 ctggagggat ggagagagaa aaacaaacag ctcctggagc agggagagtg ctggcctctt
     5641 gctctccggc tccctctgtt gccctctggt ttctccccag gttgaattcg atatcggatc
     5701 cgccaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg
     5761 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     5821 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     5881 cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt
     5941 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
     6001 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     6061 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     6121 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga
     6181 ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta
     6241 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta
     6301 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     6361 gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaact
     6421 cgagaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     6481 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     6541 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     6601 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     6661 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     6721 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     6781 gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct
     6841 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     6901 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     6961 tcttcgcctc cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcggtaccc
     7021 aattcgccct atagtgagtc gtattacgcg cgcagcggcc gaccatggcc caacttgttt
     7081 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     7141 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     7201 tggatctccg gacacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     7261 actccctctc tgcgcgctcg ctcgctcact gaggccgccc gggctttgcc cgggcggcct
     7321 cagtgagcga gcgagcgcgc agctgcctgc aggcatgcaa gctgtagcca accactagaa
     7381 ctatagctag agtcctgggc gaacaaacga tgctcgcctt ccagaaaacc gaggatgcga
     7441 accacttcat ccggggtcag cac
//
