LOCUS       sequence-251700-        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-Chrna5-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7466
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     repeat_region   4..144
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4..133
                     /label=AAV2 ITR
     mobile_element  199..364
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      548..1002
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(635..654)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     844..865
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1028..1132
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1133..1201,1202..1993)
                     /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(1351..1370)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2164..2752
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2653..2672
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  3057..3281
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(3348..3881)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4051..4056,4057..4073,4074..4079))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   4419..4559
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4430..4559
                     /label=AAV2 ITR
     promoter        4584..4824
                     /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     4584..4604
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4755..4774
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4850..4925
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4965..5412
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      5731..5740
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5737..6447
                     /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(5882..5900)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6150..6169
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    6454..7042
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6507..6527)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6925..6936)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7056..7075)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7057..7075)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    7101..7235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7138..7157)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7192..7211
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
       61 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
      121 agcgagcgcg cagctgcctg caggtctgag acaataaccc tgataaatgc ttcaataatg
      181 taagctctag gagatccgaa ccagataagt gaaatctagt tccaaactat tttgtcattt
      241 ttaattttcg tattagctta cgacgctaca cccagttccc atctattttg tcactcttcc
      301 ctaaataatc cttaaaaact ccatttccac ccctcccagt tcccaactat tttgtccgcc
      361 cacagcgggg catttttctt cctgttatgt ttttaatcaa acatcctgcc aactccatgt
      421 gacaaaccgt catcttcggc tactttttct ctgtcacaga atgaaaattt ttctgtcatc
      481 tcttcgttat taatgtttgt aattgactga atatcaacgc ttatttgcag cctgaatggc
      541 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      601 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      661 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      721 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      781 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttctta
      841 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      901 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
      961 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     1021 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1081 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1141 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1201 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1261 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1321 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1381 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1441 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1501 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1561 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1621 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1681 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1741 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1801 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     1861 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1921 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1981 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2041 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2101 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2161 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2221 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2281 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2341 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2401 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2461 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2521 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2581 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2641 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2701 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2761 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2821 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2881 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     2941 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatag accagccgcg
     3001 taacctggca aaatcggtta cggttgagta ataaatggat gccctgcgta agcgggtgtg
     3061 ggcggacaat aaagtcttaa actgaacaaa atagatctaa actatgacaa taaagtctta
     3121 aactagacag aatagttgta aactgaaatc agtccagtta tgctgtgaaa aagcatactg
     3181 gacttttgtt atggctaaag caaactcttc attttctgaa gtgcaaattg cccgtcgtat
     3241 taaagagggg cgtggccaag ggcatggtaa agactatatt cgcggcgttg tgacaattta
     3301 ccgaacaact ccgcggccgg gaagccgatc tcggcttgaa cgaattgtta ggtggcggta
     3361 cttgggtcga tatcaaagtg catcacttct tcccgtatgc ccaactttgt atagagagcc
     3421 actgcgggat cgtcaccgta atctgcttgc acgtagatca cataagcacc aagcgcgttg
     3481 gcctcatgct tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg gtgctcgccg
     3541 gagactgcga gatcatagat atagatctca ctacgcggct gctcaaactt gggcagaacg
     3601 taagccgcga gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc gatgaatgtc
     3661 ttactacgga gcaagttccc gaggtaatcg gagtccggct gatgttggga gtaggtggct
     3721 acgtctccga actcacgacc gaaaagatca agagcagccc gcatggattt gacttggtca
     3781 gggccgagcc tacatgtgcg aatgatgccc atacttgagc cacctaactt tgttttaggg
     3841 cgactgccct gctgcgtaac atcgttgctg ctccataaca tcaaacatcg acccacggcg
     3901 taacgcgctt gctgcttgga tgcccgaggc atagactgta caaaaaaaca gtcataacaa
     3961 gccatgaaaa ccgccactgc gccgttacca ccgctgcgtt cggtcaaggt tctggaccag
     4021 ttgcgtgagc gcatacgcta cttgcattac agtttacgaa ccgaacaggc ttatgtcaac
     4081 tgggttcgtg ccttcatccg tttccacggt gtgcgtcacc cggcaacctt gggcagcagc
     4141 gaagtcgagg catttctgtc ctggctggcg aacgagcgca aggtttcggt ctccacgcat
     4201 cgtcaggcat tggcggcctt gctgttcttc tacggcaagg tgctgtgcac ggatctgccc
     4261 tggcttcagg agatcggaag acctcggccg tcgcggcgct tgccggtggt gctgaccccg
     4321 gatgaagtgg ttcgcatcct cggttttctg gaaggcgagc atcgtttgtt cgcccaggac
     4381 tctagctata gttctagtgg ttggctacag cttgcatgcc tgcaggcagc tgcgcgctcg
     4441 ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc
     4501 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg
     4561 cggccgcacg cgtccccttc accgagggcc tatttcccat gattccttca tatttgcata
     4621 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     4681 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     4741 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     4801 ctttatatat cttgtggaaa ggacgaaact gaaaagtggg ttcgtcctgg ttttagagct
     4861 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     4921 ggtgcttttt tactagttct agactgcaga gggccctgcg tatgagtgca agtgggtttt
     4981 aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga cccactggac
     5041 aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg gaggggaaac
     5101 aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt gccttcgccc
     5161 ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc
     5221 ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc cgccggccca
     5281 gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc
     5341 ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc
     5401 ctgagagcgc agtcgaattc aagctgctag ctagtaccgg ttcgaacagg taagcgcccc
     5461 taaaatccct ttgggcacaa tgtgtcctga ggggagaggc agcgacctgt agatgggacg
     5521 ggggcactaa ccctcaggtt tggggcttct gaatgtgagt atcgccatgt aagcccagta
     5581 tttggccaat ctcagaaagc tcctggtccc tggagggatg gagagagaaa aacaaacagc
     5641 tcctggagca gggagagtgc tggcctcttg ctctccggct ccctctgttg ccctctggtt
     5701 tctccccagg ttgaattcga tatcggatcc gccaccatgg tgagcaaggg cgaggaggat
     5761 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     5821 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     5881 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     5941 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     6001 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     6061 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     6121 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     6181 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     6241 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     6301 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     6361 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     6421 ggcggcatgg acgagctgta caagtaactc gagaatcaac ctctggatta caaaatttgt
     6481 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     6541 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     6601 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     6661 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     6721 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     6781 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     6841 tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc
     6901 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     6961 ctgctgccgg ctctgcggcc tcttccgcgt cttcgcctcc gccctcagac gagtcggatc
     7021 tccctttggg ccgcctcccc gcggtaccca attcgcccta tagtgagtcg tattacgcgc
     7081 gcagcggccg accatggccc aacttgttta ttgcagctta taatggttac aaataaagca
     7141 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     7201 ccaaactcat caatgtatct tatcatgtct ggatctccgg acacgtgcgg accgagcggc
     7261 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     7321 aggccgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     7381 ggcatgcaag ctgtagccaa ccactagaac tatagctaga gtcctgggcg aacaaacgat
     7441 gctcgccttc cagaaaaccg aggatg
//
