LOCUS       sequence-334741-        5226 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 5226)
  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 5226)
  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-GluA2 TKIT
FEATURES             Location/Qualifiers
     source          1..5226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   231..371
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   231..360
                     /label=AAV2 ITR
     rep_origin      446..901
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(533..552)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     743..764
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(918..937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1037..1059
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1097..1115)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1183..1287
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1288..1356,1357..2148)
                     /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(1506..1525)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2319..2907
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2808..2827
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2969..3098
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             3456..4166
                     /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(3483..3511)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4106..4126
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        4515..4763
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        4785..4860
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4951..5199
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        join(5221..5226,1..70)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
       61 gagtcggtgc ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
      121 tttagcgcgt gcgccaattc tgcagacaaa tggctctaaa caacaacaat tgcattcatt
      181 ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcggccgc aggaacccct
      241 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
      301 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
      361 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
      421 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
      481 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
      541 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      601 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      661 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
      721 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
      781 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
      841 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
      901 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
      961 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     1021 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     1081 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     1141 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1201 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1261 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1321 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1381 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1441 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1501 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     1561 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1621 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1681 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1741 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1801 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     1861 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     1921 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     1981 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2041 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2101 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2161 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2221 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2281 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2341 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2401 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2461 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2521 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2581 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2641 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2701 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2761 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2821 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2881 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2941 gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact
     3001 gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     3061 cagagaggga gtggccaact ccatcactag gggttcctgc ggccgctgca gagggcccgt
     3121 cgacacattt ccccgactcg agaattctga cgtcattatt atcagggcct ctattgtgtc
     3181 aaaaggcata cctgggagat aaggattctt ctgccttcac ttcgtggttt tagcagctcc
     3241 ttgctaaatt tcgacctcaa aatgcagagg atctaatttg ctgaggaaaa cggtcaaaga
     3301 aggaaaagga ggaaagggaa acgaggggat attttgtgga tgctctactt ttcttggaaa
     3361 tgcaaaagat tatgcatatt tctgtcctcc tttctcctgt tttatgggga ctgatttttg
     3421 gtgtctcttc taacactagt ggtggcggtg gctcgagtaa aggagaagaa cttttcactg
     3481 gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca
     3541 gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta
     3601 ctggaaaact acctgttcct tggccaacac ttgtcactac tttaacttat ggtgttcaat
     3661 gcttttcaag atacccagat catatgaaac ggcatgactt tttcaagagt gccatgcccg
     3721 aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac aagacacgtg
     3781 ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt
     3841 ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac gatcaccagg
     3901 tgtacatcat ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa attagacaca
     3961 acattgaaga tggaggcgtt caactagcag accattatca acaaaatact ccaattggcg
     4021 atgggcccgt ccttttacca gacaaccatt acctgtttac aacttctact ctttcgaaag
     4081 atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta
     4141 cacatggcat ggatgaacta tacaaaggtg gcggtggctc gggcggaggt gggtcgggtg
     4201 gcggcggatc aggcggtggc ggatccagca tacagatagg taggtacctt ttgtgttatg
     4261 cctttgaatt gtgcataatt tggtggtaat ctttgttcac agtgtgcaaa ttaattcatg
     4321 tcatgtagac ttatgtcata ccttgactgc attccagatt gaaactacct agaatataca
     4381 tgattttctt aggctgaagc aaaaagaaat gactatgcct tttgacacaa cccaggttta
     4441 gctggtggct gttccatcat ggtttttacc tcctgaattc ggatccctcg agcgctggcc
     4501 ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca
     4561 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     4621 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     4681 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     4741 tcttgtggaa aggacgaaac accgaacagc caccagctaa acctgtttta gagctagaaa
     4801 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     4861 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt
     4921 gcgccaattc tgcagacaaa tggctctagc gagggcctat ttcccatgat tccttcatat
     4981 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     5041 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     5101 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     5161 ttcttggctt tatatatctt gtggaaagga cgaaacaccg tatgcctttt gacacaatag
     5221 gtttta
//
