LOCUS       sequence-250831-        7417 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 7417)
  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 7417)
  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-Chrna3-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7417
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     mobile_element  169..334
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      518..972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(605..624)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     814..835
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        998..1102
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1103..1171,1172..1963)
                     /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(1321..1340)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2134..2722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2623..2642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  3027..3251
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(3318..3851)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4021..4026,4027..4043,4044..4049))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   4389..4529
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4400..4529
                     /label=AAV2 ITR
     promoter        4554..4794
                     /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     4554..4574
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4725..4744
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4820..4895
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4935..5382
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      5701..5710
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5707..6417
                     /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(5852..5870)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6120..6139
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    6424..7012
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6477..6497)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6895..6906)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7026..7045)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7027..7045)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    7071..7205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7108..7127)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7162..7181
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
       61 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggtctgag
      121 acaataaccc tgataaatgc ttcaataatg taagctctag gagatccgaa ccagataagt
      181 gaaatctagt tccaaactat tttgtcattt ttaattttcg tattagctta cgacgctaca
      241 cccagttccc atctattttg tcactcttcc ctaaataatc cttaaaaact ccatttccac
      301 ccctcccagt tcccaactat tttgtccgcc cacagcgggg catttttctt cctgttatgt
      361 ttttaatcaa acatcctgcc aactccatgt gacaaaccgt catcttcggc tactttttct
      421 ctgtcacaga atgaaaattt ttctgtcatc tcttcgttat taatgtttgt aattgactga
      481 atatcaacgc ttatttgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
      541 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
      601 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
      661 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
      721 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
      781 cgccctttga cgttggagtc cacgttctta atagtggact cttgttccaa actggaacaa
      841 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
      901 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
      961 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1021 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1081 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     1141 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     1201 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     1261 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     1321 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     1381 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1441 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     1501 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     1561 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     1621 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     1681 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     1741 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     1801 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     1861 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     1921 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     1981 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     2041 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2101 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2161 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2221 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2281 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2341 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2401 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2461 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2521 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2581 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2641 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2701 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2761 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     2821 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2881 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     2941 cggtatttca caccgcatag accagccgcg taacctggca aaatcggtta cggttgagta
     3001 ataaatggat gccctgcgta agcgggtgtg ggcggacaat aaagtcttaa actgaacaaa
     3061 atagatctaa actatgacaa taaagtctta aactagacag aatagttgta aactgaaatc
     3121 agtccagtta tgctgtgaaa aagcatactg gacttttgtt atggctaaag caaactcttc
     3181 attttctgaa gtgcaaattg cccgtcgtat taaagagggg cgtggccaag ggcatggtaa
     3241 agactatatt cgcggcgttg tgacaattta ccgaacaact ccgcggccgg gaagccgatc
     3301 tcggcttgaa cgaattgtta ggtggcggta cttgggtcga tatcaaagtg catcacttct
     3361 tcccgtatgc ccaactttgt atagagagcc actgcgggat cgtcaccgta atctgcttgc
     3421 acgtagatca cataagcacc aagcgcgttg gcctcatgct tgaggagatt gatgagcgcg
     3481 gtggcaatgc cctgcctccg gtgctcgccg gagactgcga gatcatagat atagatctca
     3541 ctacgcggct gctcaaactt gggcagaacg taagccgcga gagcgccaac aaccgcttct
     3601 tggtcgaagg cagcaagcgc gatgaatgtc ttactacgga gcaagttccc gaggtaatcg
     3661 gagtccggct gatgttggga gtaggtggct acgtctccga actcacgacc gaaaagatca
     3721 agagcagccc gcatggattt gacttggtca gggccgagcc tacatgtgcg aatgatgccc
     3781 atacttgagc cacctaactt tgttttaggg cgactgccct gctgcgtaac atcgttgctg
     3841 ctccataaca tcaaacatcg acccacggcg taacgcgctt gctgcttgga tgcccgaggc
     3901 atagactgta caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc gccgttacca
     3961 ccgctgcgtt cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta cttgcattac
     4021 agtttacgaa ccgaacaggc ttatgtcaac tgggttcgtg ccttcatccg tttccacggt
     4081 gtgcgtcacc cggcaacctt gggcagcagc gaagtcgagg catttctgtc ctggctggcg
     4141 aacgagcgca aggtttcggt ctccacgcat cgtcaggcat tggcggcctt gctgttcttc
     4201 tacggcaagg tgctgtgcac ggatctgccc tggcttcagg agatcggaag acctcggccg
     4261 tcgcggcgct tgccggtggt gctgaccccg gatgaagtgg ttcgcatcct cggttttctg
     4321 gaaggcgagc atcgtttgtt cgcccaggac tctagctata gttctagtgg ttggctacag
     4381 cttgcatgcc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
     4441 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
     4501 agtggccaac tccatcacta ggggttcctg cggccgcacg cgtccccttc accgagggcc
     4561 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     4621 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     4681 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     4741 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaacg
     4801 aaaccctctg actaccaagg ttttagagct agaaatagca agttaaaata aggctagtcc
     4861 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tactagttct agactgcaga
     4921 gggccctgcg tatgagtgca agtgggtttt aggaccagga tgaggcgggg tgggggtgcc
     4981 tacctgacga ccgaccccga cccactggac aagcacccaa cccccattcc ccaaattgcg
     5041 catcccctat cagagagggg gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca
     5101 gcttcagcac cgcggacagt gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc
     5161 agcactgaag gcgcgctgac gtcactcgcc ggtcccccgc aaactcccct tcccggccac
     5221 cttggtcgcg tccgcgccgc cgccggccca gccggaccgc accacgcgag gcgcgagata
     5281 ggggggcacg ggcgcgacca tctgcgctgc ggcgccggcg actcagcgct gcctcagtct
     5341 gcggtgggca gcggaggagt cgtgtcgtgc ctgagagcgc agtcgaattc aagctgctag
     5401 ctagtaccgg ttcgaacagg taagcgcccc taaaatccct ttgggcacaa tgtgtcctga
     5461 ggggagaggc agcgacctgt agatgggacg ggggcactaa ccctcaggtt tggggcttct
     5521 gaatgtgagt atcgccatgt aagcccagta tttggccaat ctcagaaagc tcctggtccc
     5581 tggagggatg gagagagaaa aacaaacagc tcctggagca gggagagtgc tggcctcttg
     5641 ctctccggct ccctctgttg ccctctggtt tctccccagg ttgaattcga tatcggatcc
     5701 gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     5761 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     5821 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     5881 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     5941 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     6001 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     6061 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     6121 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     6181 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     6241 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     6301 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     6361 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaactc
     6421 gagaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     6481 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     6541 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     6601 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     6661 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     6721 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     6781 ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg
     6841 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     6901 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     6961 cttcgcctcc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcggtaccca
     7021 attcgcccta tagtgagtcg tattacgcgc gcagcggccg accatggccc aacttgttta
     7081 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     7141 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     7201 ggatctccgg acacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     7261 ctccctctct gcgcgctcgc tcgctcactg aggccgcccg ggctttgccc gggcggcctc
     7321 agtgagcgag cgagcgcgca gctgcctgca ggcatgcaag ctgtagccaa ccactagaac
     7381 tatagctaga gtcctgggcg aacaaacgat gctcgcc
//
