LOCUS       sequence-250827-        7492 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAAV encoding gRNA sequence for loss-of-function indels.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7492)
  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 7492)
  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-Chrna7-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7492
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        166..406
                     /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     166..186
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     337..356
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        432..507
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        547..994
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1313..1322
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1319..2029
                     /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(1464..1482)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1732..1751
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2036..2624
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2089..2109)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2507..2518)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2638..2657)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2639..2657)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2683..2817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2720..2739)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2774..2793
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3078..3218
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3078..3207
                     /label=AAV2 ITR
     mobile_element  3273..3438
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      3622..4076
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3709..3728)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3918..3939
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4102..4206
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4207..4275,4276..5067)
                     /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(4425..4444)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5238..5826
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5727..5746
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  6131..6355
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(6422..6955)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7125..7130,7131..7147,7148..7153))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtcccct tcaccgaggg cctatttccc
      181 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
      241 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
      301 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
      361 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa cggtgagcgg
      421 ctgcgagtcg tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      481 cttgaaaaag tggcaccgag tcggtgcttt tttactagtt ctagactgca gagggccctg
      541 cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac
      601 gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct
      661 atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc
      721 accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga
      781 aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg
      841 cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca
      901 cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg
      961 cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaat tcaagctgct agctagtacc
     1021 ggttcgaaca ggtaagcgcc cctaaaatcc ctttgggcac aatgtgtcct gaggggagag
     1081 gcagcgacct gtagatggga cgggggcact aaccctcagg tttggggctt ctgaatgtga
     1141 gtatcgccat gtaagcccag tatttggcca atctcagaaa gctcctggtc cctggaggga
     1201 tggagagaga aaaacaaaca gctcctggag cagggagagt gctggcctct tgctctccgg
     1261 ctccctctgt tgccctctgg tttctcccca ggttgaattc gatatcggat ccgccaccat
     1321 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     1381 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     1441 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     1501 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     1561 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     1621 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     1681 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     1741 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     1801 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     1861 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     1921 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     1981 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaac tcgagaatca
     2041 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     2101 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     2161 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     2221 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     2281 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     2341 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     2401 cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg
     2461 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     2521 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     2581 ccgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcggtacc caattcgccc
     2641 tatagtgagt cgtattacgc gcgcagcggc cgaccatggc ccaacttgtt tattgcagct
     2701 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2761 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctcc
     2821 ggacacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     2881 ctgcgcgctc gctcgctcac tgaggccgcc cgggctttgc ccgggcggcc tcagtgagcg
     2941 agcgagcgcg cagctgcctg caggcatgca agctgtagcc aaccactaga actatagcta
     3001 gagtcctggg cgaacaaacg atgctcgcct tccagaaaac cgagsatgcv aacggtgaag
     3061 gggacgcgtg cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     3121 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     3181 ggcctcagtg agcgagcgag cgcgcagctg cctgcaggtc tgagacaata accctgataa
     3241 atgcttcaat aatgtaagct ctaggagatc cgaaccagat aagtgaaatc tagttccaaa
     3301 ctattttgtc atttttaatt ttcgtattag cttacgacgc tacacccagt tcccatctat
     3361 tttgtcactc ttccctaaat aatccttaaa aactccattt ccacccctcc cagttcccaa
     3421 ctattttgtc cgcccacagc ggggcatttt tcttcctgtt atgtttttaa tcaaacatcc
     3481 tgccaactcc atgtgacaaa ccgtcatctt cggctacttt ttctctgtca cagaatgaaa
     3541 atttttctgt catctcttcg ttattaatgt ttgtaattga ctgaatatca acgcttattt
     3601 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3661 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3721 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3781 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3841 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3901 agtccacgtt cttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3961 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4021 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     4081 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4141 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4201 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     4261 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     4321 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     4381 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     4441 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     4501 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4561 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4621 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4681 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4741 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4801 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4861 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4921 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4981 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     5041 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     5101 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5161 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5221 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5281 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5341 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5401 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5461 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5521 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5581 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5641 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5701 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5761 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5821 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5881 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5941 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     6001 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     6061 atagaccagc cgcgtaacct ggcaaaatcg gttacggttg agtaataaat ggatgccctg
     6121 cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa caaaatagat ctaaactatg
     6181 acaataaagt cttaaactag acagaatagt tgtaaactga aatcagtcca gttatgctgt
     6241 gaaaaagcat actggacttt tgttatggct aaagcaaact cttcattttc tgaagtgcaa
     6301 attgcccgtc gtattaaaga ggggcgtggc caagggcatg gtaaagacta tattcgcggc
     6361 gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc gatctcggct tgaacgaatt
     6421 gttaggtggc ggtacttggg tcgatatcaa agtgcatcac ttcttcccgt atgcccaact
     6481 ttgtatagag agccactgcg ggatcgtcac cgtaatctgc ttgcacgtag atcacataag
     6541 caccaagcgc gttggcctca tgcttgagga gattgatgag cgcggtggca atgccctgcc
     6601 tccggtgctc gccggagact gcgagatcat agatatagat ctcactacgc ggctgctcaa
     6661 acttgggcag aacgtaagcc gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa
     6721 gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta atcggagtcc ggctgatgtt
     6781 gggagtaggt ggctacgtct ccgaactcac gaccgaaaag atcaagagca gcccgcatgg
     6841 atttgacttg gtcagggccg agcctacatg tgcgaatgat gcccatactt gagccaccta
     6901 actttgtttt agggcgactg ccctgctgcg taacatcgtt gctgctccat aacatcaaac
     6961 atcgacccac ggcgtaacgc gcttgctgct tggatgcccg aggcatagac tgtacaaaaa
     7021 aacagtcata acaagccatg aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca
     7081 aggttctgga ccagttgcgt gagcgcatac gctacttgca ttacagttta cgaaccgaac
     7141 aggcttatgt caactgggtt cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa
     7201 ccttgggcag cagcgaagtc gaggcatttc tgtcctggct ggcgaacgag cgcaaggttt
     7261 cggtctccac gcatcgtcag gcattggcgg ccttgctgtt cttctacggc aaggtgctgt
     7321 gcacggatct gccctggctt caggagatcg gaagacctcg gccgtcgcgg cgcttgccgg
     7381 tggtgctgac cccggatgaa gtggttcgca tcctcggttt tctggaaggc gagcatcgtt
     7441 tgttcgccca ggactctagc tatagttcta gtggttggct acagcttgca tg
//
