LOCUS       sequence-245821-        7541 bp    DNA     circular SYN 15-APR-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 7541)
  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 7541)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..7541
                     /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..4327
                     /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     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        4468..4915
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      5221..5230
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5227..5937
                     /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(5372..5390)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5640..5659
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    5944..6532
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5997..6017)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6415..6426)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6546..6565)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6547..6565)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    6591..6725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   6755..6895
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6755..6884
                     /label=AAV2 ITR
     polyA_signal    7032..7166
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     mobile_element  7195..7360
                     /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 acgaaaccct ctgactacca aggttttaga gctagaaata gcaagttaaa
     4381 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttactagt
     4441 tctagactgc agagggccct gcgtatgagt gcaagtgggt tttaggacca ggatgaggcg
     4501 gggtgggggt gcctacctga cgaccgaccc cgacccactg gacaagcacc caacccccat
     4561 tccccaaatt gcgcatcccc tatcagagag ggggagggga aacaggatgc ggcgaggcgc
     4621 gtgcgcactg ccagcttcag caccgcggac agtgccttcg cccccgcctg gcggcgcgcg
     4681 ccaccgccgc ctcagcactg aaggcgcgct gacgtcactc gccggtcccc cgcaaactcc
     4741 ccttcccggc caccttggtc gcgtccgcgc cgccgccggc ccagccggac cgcaccacgc
     4801 gaggcgcgag ataggggggc acgggcgcga ccatctgcgc tgcggcgccg gcgactcagc
     4861 gctgcctcag tctgcggtgg gcagcggagg agtcgtgtcg tgcctgagag cgcagtcgag
     4921 aattccaccg gttcgaacag gtaagcgccc ctaaaatccc tttggcacaa tgtgtcctga
     4981 ggggagaggc agcgacctgt agatgggacg ggggcactaa ccctcagggt ttggggttct
     5041 gaatgtgagt atcgccatgt aagcccagta tttggccaat ctcagaaagc tcctggctcc
     5101 ctggaggatg gagagagaaa aacaaacagc tcctggagca gggagagtgc tggcctcttg
     5161 ctctccggct ccctctgttg ccctctggtt tctccccagg ttgaattcga tatcggatcc
     5221 gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     5281 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     5341 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     5401 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     5461 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     5521 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     5581 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     5641 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     5701 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     5761 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     5821 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     5881 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaactc
     5941 gagaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     6001 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     6061 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     6121 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     6181 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     6241 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     6301 ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg
     6361 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     6421 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     6481 cttcgcctcc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcggtaccca
     6541 attcgcccta tagtgagtcg tattacgcgc gcagcggccg accatggccc aacttgttta
     6601 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     6661 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     6721 ggatctccgg accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     6781 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     6841 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggaagct
     6901 gtaagcttgt cgagaagtac tagaggatca taatcagcca taccacattt gtagaggttt
     6961 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     7021 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     7081 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     7141 tcaatgtatc ttatcatgtc tggatctgat cactgatatc gcctaggaga tccgaaccag
     7201 ataagtgaaa tctagttcca aactattttg tcatttttaa ttttcgtatt agcttacgac
     7261 gctacaccca gttcccatct attttgtcac tcttccctaa ataatcctta aaaactccat
     7321 ttccacccct cccagttccc aactattttg tccgcccaca gcggggcatt tttcttcctg
     7381 ttatgttttt aatcaaacat cctgccaact ccatgtgaca aaccgtcatc ttcggctact
     7441 ttttctctgt cacagaatga aaatttttct gtcatctctt cgttattaat gtttgtaatt
     7501 gactgaatat caacgcttat ttgcagcctg aatggcgaat g
//
