LOCUS       sequence-335504-        5056 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 2 guides + donor DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5056)
  AUTHORS   Fang H, Bygrave AM, Roth RH, Johnson RC, Huganir RL
  TITLE     An optimized CRISPR/Cas9 approach for precise genome editing in 
            neurons.
  JOURNAL   Elife. 2021 Mar 10;10. pii: 65202. doi: 10.7554/eLife.65202.
  PUBMED    33689678
REFERENCE   2  (bases 1 to 5056)
  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: "Elife."; 
            date: "2021-03-10"; volume: "10. pii: 65202. doi"; pages: " 
            10.7554/eLife.65202"
COMMENT     Sequence Label: pAAV-SEP-GluA1 TKIT
FEATURES             Location/Qualifiers
     source          1..5056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             345..1055
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(372..400)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     995..1015
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        1377..1625
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1646..1721
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1812..2060
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2082..2157
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   2319..2459
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2319..2448
                     /label=AAV2 ITR
     rep_origin      2534..2989
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2621..2640)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2831..2852
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3006..3025)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3125..3147
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3185..3203)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3271..3375
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3376..3444,3445..4236)
                     /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(3594..3613)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4407..4995
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4896..4915
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgctg cagagggccc gtcgacacat ttccccgact cgagaattct
      181 gacgtcatta ttatcagggc cttcctggtg ttccattgct aaggttggac cagggcttct
      241 ttttcgccaa tgtaaaaagg aatatgccgt acatctttgc ctttttctgc accggttttc
      301 taggtgcggt tgtgggtgcc aattctagtg gtggcggtgg ctcgagtaaa ggagaagaac
      361 ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat
      421 tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta
      481 tttgcactac tggaaaacta cctgttcctt ggccaacact tgtcactact ttaacttatg
      541 gtgttcaatg cttttcaaga tacccagatc atatgaaacg gcatgacttt ttcaagagtg
      601 ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca
      661 agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag
      721 gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac aactataacg
      781 atcaccaggt gtacatcatg gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa
      841 ttagacacaa cattgaagat ggaggcgttc aactagcaga ccattatcaa caaaatactc
      901 caattggcga tgggcccgtc cttttaccag acaaccatta cctgtttaca acttctactc
      961 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
     1021 ctgggattac acatggcatg gatgaactat acaaaggtgg cggtggctcg ggcggaggtg
     1081 ggtcgggtgg cggcggatca ggcggtggcg gatccttccc caacaatatc cagataggtg
     1141 agtgagaggg gcagctgggg agggactttg gggatctggc caggattttc tttttcgttt
     1201 ttttcttttt cttttttctt tttttttatt tggaggggag atttctcaac ccattctcag
     1261 tcggagctgt tgtgcttagc aatggaacac caccaaccat agatttggtc ttcccacatc
     1321 atggttttta cctcctgaat tcggatccct cgagcgctgg ccttttgctc acatgtgagg
     1381 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     1441 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     1501 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     1561 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     1621 acaccgggaa gaccaaatct atggtgtttt agagctagaa atagcaagtt aaaataaggc
     1681 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag
     1741 aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa
     1801 atggctctag cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     1861 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     1921 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     1981 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     2041 tgtggaaagg acgaaacacc gttagcaatg gaacaccagg agttttagag ctagaaatag
     2101 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     2161 tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg
     2221 ccaattctgc agacaaatgg ctctagaaca acaacaattg cattcatttt atgtttcagg
     2281 ttcaggggga gatgtgggag gttttttaaa gcggccgcag gaacccctag tgatggagtt
     2341 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     2401 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     2461 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     2521 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2581 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     2641 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2701 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     2761 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2821 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     2881 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     2941 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     3001 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     3061 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3121 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     3181 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     3241 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3301 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3361 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3421 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3481 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3541 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3601 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3661 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3721 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3781 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3841 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3901 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3961 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4021 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4081 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4141 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4201 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4261 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4321 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4381 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4441 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4501 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     4561 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4621 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4681 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4741 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4801 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4861 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4921 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4981 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5041 ccttttgctc acatgt
//
