LOCUS       sequence-251702-        7493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAAV encoding control gRNA for CRISPR gRNAs listed above.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7493)
  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 7493)
  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-control-gRNA-hSyn-mCherry-WPRE-SV40pA
FEATURES             Location/Qualifiers
     source          1..7493
                     /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   3079..3219
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3079..3208
                     /label=AAV2 ITR
     mobile_element  3274..3439
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      3623..4077
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3710..3729)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3919..3940
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4103..4207
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4208..4276,4277..5068)
                     /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(4426..4445)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5239..5827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5728..5747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  6132..6356
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(6423..6956)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7126..7131,7132..7148,7149..7154))
                     /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 cgcgaggtat
      421 tcggctccgc ggttttagag 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 cgaggatgcg aaccacttca
     3061 tgggacgcgt gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     3121 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     3181 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggt ctgagacaat aaccctgata
     3241 aatgcttcaa taatgtaagc tctaggagat ccgaaccaga taagtgaaat ctagttccaa
     3301 actattttgt catttttaat tttcgtatta gcttacgacg ctacacccag ttcccatcta
     3361 ttttgtcact cttccctaaa taatccttaa aaactccatt tccacccctc ccagttccca
     3421 actattttgt ccgcccacag cggggcattt ttcttcctgt tatgttttta atcaaacatc
     3481 ctgccaactc catgtgacaa accgtcatct tcggctactt tttctctgtc acagaatgaa
     3541 aatttttctg tcatctcttc gttattaatg tttgtaattg actgaatatc aacgcttatt
     3601 tgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3661 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3721 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3781 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3841 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3901 gagtccacgt tcttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3961 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4021 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     4081 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4141 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4201 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4261 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4321 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4381 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4441 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4501 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4561 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4621 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4681 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4741 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4801 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4861 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4921 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4981 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5041 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5101 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5161 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5221 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5281 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5341 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5401 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5461 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5521 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5581 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5641 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5701 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5761 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5821 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5881 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5941 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6001 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6061 catagaccag ccgcgtaacc tggcaaaatc ggttacggtt gagtaataaa tggatgccct
     6121 gcgtaagcgg gtgtgggcgg acaataaagt cttaaactga acaaaataga tctaaactat
     6181 gacaataaag tcttaaacta gacagaatag ttgtaaactg aaatcagtcc agttatgctg
     6241 tgaaaaagca tactggactt ttgttatggc taaagcaaac tcttcatttt ctgaagtgca
     6301 aattgcccgt cgtattaaag aggggcgtgg ccaagggcat ggtaaagact atattcgcgg
     6361 cgttgtgaca atttaccgaa caactccgcg gccgggaagc cgatctcggc ttgaacgaat
     6421 tgttaggtgg cggtacttgg gtcgatatca aagtgcatca cttcttcccg tatgcccaac
     6481 tttgtataga gagccactgc gggatcgtca ccgtaatctg cttgcacgta gatcacataa
     6541 gcaccaagcg cgttggcctc atgcttgagg agattgatga gcgcggtggc aatgccctgc
     6601 ctccggtgct cgccggagac tgcgagatca tagatataga tctcactacg cggctgctca
     6661 aacttgggca gaacgtaagc cgcgagagcg ccaacaaccg cttcttggtc gaaggcagca
     6721 agcgcgatga atgtcttact acggagcaag ttcccgaggt aatcggagtc cggctgatgt
     6781 tgggagtagg tggctacgtc tccgaactca cgaccgaaaa gatcaagagc agcccgcatg
     6841 gatttgactt ggtcagggcc gagcctacat gtgcgaatga tgcccatact tgagccacct
     6901 aactttgttt tagggcgact gccctgctgc gtaacatcgt tgctgctcca taacatcaaa
     6961 catcgaccca cggcgtaacg cgcttgctgc ttggatgccc gaggcataga ctgtacaaaa
     7021 aaacagtcat aacaagccat gaaaaccgcc actgcgccgt taccaccgct gcgttcggtc
     7081 aaggttctgg accagttgcg tgagcgcata cgctacttgc attacagttt acgaaccgaa
     7141 caggcttatg tcaactgggt tcgtgccttc atccgtttcc acggtgtgcg tcacccggca
     7201 accttgggca gcagcgaagt cgaggcattt ctgtcctggc tggcgaacga gcgcaaggtt
     7261 tcggtctcca cgcatcgtca ggcattggcg gccttgctgt tcttctacgg caaggtgctg
     7321 tgcacggatc tgccctggct tcaggagatc ggaagacctc ggccgtcgcg gcgcttgccg
     7381 gtggtgctga ccccggatga agtggttcgc atcctcggtt ttctggaagg cgagcatcgt
     7441 ttgttcgccc aggactctag ctatagttct agtggttggc tacagcttgc atg
//
