LOCUS       sequence-245826-        7540 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 7540)
  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 7540)
  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-beta2 RD-1-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..457
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     299..320
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        484..588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(589..657,658..1449)
                     /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(807..826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1620..2208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2109..2128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  2511..2735
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2802..3335)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3524..3529,3530..3546,3547..3552))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   3922..4062
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3933..4062
                     /label=AAV2 ITR
     promoter        4087..4335
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4087..4107
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4258..4277
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4353..4428
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4467..4914
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      5220..5229
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5226..5936
                     /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(5371..5389)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5639..5658
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    5943..6531
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5996..6016)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6414..6425)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6545..6564)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6546..6564)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    6590..6724
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   6754..6894
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6754..6883
                     /label=AAV2 ITR
     polyA_signal    7031..7165
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     mobile_element  7194..7359
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
      301 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
      361 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
      421 aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat
      481 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
      541 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
      601 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
      661 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
      721 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
      781 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
      841 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
      901 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
      961 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     1021 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     1081 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     1141 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     1201 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     1261 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     1321 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     1381 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     1441 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     1501 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     1561 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1621 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1681 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1741 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1801 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1861 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1921 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1981 tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg
     2041 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2101 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2161 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2221 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2281 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2341 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     2401 cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct
     2461 ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga
     2521 caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag
     2581 acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt
     2641 tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga
     2701 ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac
     2761 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     2821 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     2881 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     2941 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3001 gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc
     3061 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3121 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3181 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3241 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3301 ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca
     3361 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa
     3421 acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa
     3481 ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca
     3541 ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac
     3601 cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc
     3661 ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg
     3721 cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt
     3781 ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt
     3841 gttcgcccag gactctagct atagttctag tggttggcta cattattgaa gcatttatca
     3901 gggttattgt ctcagagcat gcctgcaggc agctgcgcgc tcgctcgctc actgaggccg
     3961 cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
     4021 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgtcccc
     4081 ttcaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     4141 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     4201 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     4261 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     4321 aaaggacgaa acatcagctt gttatagcgg gagttttaga gctagaaata gcaagttaaa
     4381 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttactagt
     4441 ctagactgca gagggccctg cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg
     4501 ggtgggggtg cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt
     4561 ccccaaattg cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg
     4621 tgcgcactgc cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc
     4681 caccgccgcc tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc
     4741 cttcccggcc accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg
     4801 aggcgcgaga taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg
     4861 ctgcctcagt ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga
     4921 attccaccgg ttcgaacagg taagcgcccc taaaatccct ttggcacaat gtgtcctgag
     4981 gggagaggca gcgacctgta gatgggacgg gggcactaac cctcagggtt tggggttctg
     5041 aatgtgagta tcgccatgta agcccagtat ttggccaatc tcagaaagct cctggctccc
     5101 tggaggatgg agagagaaaa acaaacagct cctggagcag ggagagtgct ggcctcttgc
     5161 tctccggctc cctctgttgc cctctggttt ctccccaggt tgaattcgat atcggatccg
     5221 ccaccatggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     5281 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     5341 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     5401 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     5461 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     5521 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     5581 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     5641 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     5701 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     5761 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     5821 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     5881 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaactcg
     5941 agaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     6001 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     6061 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     6121 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     6181 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     6241 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     6301 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     6361 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     6421 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     6481 ttcgcctccg ccctcagacg agtcggatct ccctttgggc cgcctccccg cggtacccaa
     6541 ttcgccctat agtgagtcgt attacgcgcg cagcggccga ccatggccca acttgtttat
     6601 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     6661 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     6721 gatctccgga ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     6781 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     6841 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggaagctg
     6901 taagcttgtc gagaagtact agaggatcat aatcagccat accacatttg tagaggtttt
     6961 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     7021 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     7081 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     7141 caatgtatct tatcatgtct ggatctgatc actgatatcg cctaggagat ccgaaccaga
     7201 taagtgaaat ctagttccaa actattttgt catttttaat tttcgtatta gcttacgacg
     7261 ctacacccag ttcccatcta ttttgtcact cttccctaaa taatccttaa aaactccatt
     7321 tccacccctc ccagttccca actattttgt ccgcccacag cggggcattt ttcttcctgt
     7381 tatgttttta atcaaacatc ctgccaactc catgtgacaa accgtcatct tcggctactt
     7441 tttctctgtc acagaatgaa aatttttctg tcatctcttc gttattaatg tttgtaattg
     7501 actgaatatc aacgcttatt tgcagcctga atggcgaatg
//
