LOCUS       SK0687(pAAV-GfaA        7084 bp    DNA     circular SYN 22-OCT-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7084)
  AUTHORS   Institute for Basic Science
  TITLE     Direct Submission
  JOURNAL   Exported Oct 22, 2025 from SnapGene 8.1.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     rep_origin      28..616
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     repeat_region   678..818
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     primer_bind     827..857
                     /label=MluI-gfaABC1D-JSKF
                     /note="color: black; sequence: 
                     atcgACGCGTAACATATCCTGGTGTGGAGTAGGGG"
     misc_feature    833..1513
                     /label=GfaABC1D
                     /note="color: #ff99cc"
     primer_bind     complement(1502..1519)
                     /label=gfABC1D-XbaI-JSKR
                     /note="color: black; sequence: atcgTCTAGAGCGAGCAGCGGA"
     primer_bind     1539..1601
                     /label=HA-CRY2(EA9,NheI)-F
                     /note="color: black; sequence: 
                     GACTGCTAGCGCCACCATGGGATACCCATACGACGTGCCTGACTACGCCCCACCGGTCA
                     TGAAGATGGAC; added: 2022-02-13"
     primer_bind     1539..1598
                     /label=FLAG-CRY2(EA9,NheI)-F
                     /note="color: black; sequence: 
                     GACTGCTAGCGCCACCATGGGAGACTACAAGGATGACGACGATAAGCCACCGGTCATGA
                     AGATG; added: 2022-02-13"
     CDS             join(1551..1556,1557..1580)
                     /codon_start=1
                     /label=FLAG tag
                     /note="This feature has 2 segments:
                        1: 1551 .. 1556 / #99ccff
                        2: 1557 .. 1580 / #99ccff"
                     /translation="MGDYKDDDDK"
     primer_bind     1589..1614
                     /label=CRY2(EA9,AgeI)-F
                     /note="color: black; sequence: 
                     GACTACCGGTCGCCACCATGAAGATGGACAAAAAGACCATCG; added: 
                     2021-08-12"
     CDS             1590..3083
                     /codon_start=1
                     /label=CRY2(E281A)
                     /note="color: #33cccc"
                     /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
                     FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
                     DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
                     ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
                     EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGA
                     ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
                     VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
                     ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
                     SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAA"
     primer_bind     complement(1634..1660)
                     /label=CRY2(seq)-NR
                     /note="color: black; sequence: GCGAGCGCGGGATTATCTTCTATTCTC;
                     added: 2022-03-20"
     primer_bind     complement(2407..2438)
                     /label=Fragment 1.REV
                     /note="color: black; sequence: 
                     GCTTTCGGCGCCTTCGCTGTTTTTATCGCGAG"
     primer_bind     complement(2407..2438)
                     /label=STIM1(E281A)-R
                     /note="color: black; sequence: 
                     GCTTTCGGCGCCTTCGCTGTTTTTATCGCGAG"
     primer_bind     2424..2453
                     /label=Fragment 2.FOR
                     /note="color: black; sequence: 
                     GAAGGCGCCGAAAGCGCCGATCTGTTCCTG"
     primer_bind     2424..2453
                     /label=PHR(E281A)-F
                     /note="color: black; sequence: 
                     GAAGGCGCCGAAAGCGCCGATCTGTTCCTG"
     primer_bind     complement(2599..2617)
                     /label=HA-CRY2(EA9)-R
                     /note="color: black; sequence: GCATCCACGAGTGGGTACC; added: 
                     2022-02-13"
     primer_bind     complement(2665..2690)
                     /label=CRY2(internal)-R
                     /note="color: black; sequence: GACAGCAAATGAGGACACGATTACCC; 
                     added: 2022-02-21"
     primer_bind     3010..3032
                     /label=CRY2(seq)-CF
                     /note="color: black; sequence: TAGATACAGCACGCGAGCTGCTG; 
                     added: 2022-03-20"
     CDS             3084..3110
                     /codon_start=1
                     /label=A9
                     /note="color: #ffffff"
                     /translation="ARDPPDLDN"
     CDS             3204..4547
                     /codon_start=1
                     /label=STIM1(238-685)
                     /note="color: #ff0000"
                     /translation="KEHMKKMMKDLEGLHRAEQSLHDLQERLHKAQEEHRTVEVEKVHL
                     EKKLRDEINLAKQEAQRLKELREGTENERSRQKYAEEELEQVREALRKAEKELESHSSW
                     YAPEALQKWLQLTHEVEVQYYNIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSS
                     SLDDVDHKILTAKQALSEVTAALRERLHRWQQIEILCGFQIVNNPGIHSLVAALNIDPS
                     WMGSTRPNPAHFIMTDDVDDMDEEIVSPLSMQSPSLQSSVRQRLTEPQHGLGSQRDLTH
                     SDSESSLHMSDRQRVAPKPPQMSRAADEALNAMTSNGSHRLIEGVHPGSLVEKLPDSPA
                     LAKKALLALNHGLDKAHSLMELSPSAPPGGSPHLDSSRSHSPSSPDPDTPSPVGDSRAL
                     QASRNTRIPHLAGKKAVAEEDNGSIGEETDSSPGRKKFPLKIFKKPLKK"
     primer_bind     complement(3852..3872)
                     /label=STIM1(~460)-stop-BamHI-JGSR
                     /note="color: black; sequence: 
                     TCAGTTATCTAGATCCGGTGGATCCCTACCAGCTGGGGTCTATGTTGAG"
     primer_bind     4556..4596
                     /label=BamHI-XhoI-CW3SL-JGSF
                     /note="color: black; sequence: 
                     TGGATGGGCAGTTAGGGATCCctcgagataatcaacctctggattacaaaatttg"
     misc_feature    4571..4997
                     /label=W3SL
                     /note="Optimization of AAV expression cassettes to improve 
                     packaging capacity and transgene expression in neurons
                     WPRE3 + Late SV40
                     "
                     /note="color: #ffffff"
     primer_bind     complement(4624..4644)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
                     /note="color: black; sequence: CATAGCGTAAAAGGAGCAACA"
     primer_bind     complement(4986..5027)
                     /label=CW3SL-RsrII-JGSR
                     /note="color: black; sequence: 
                     tcctgcggccgctcggtccgtcctgcggccgctttaaaaaac"
     repeat_region   5024..5164
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     rep_origin      5239..5694
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        5976..6080
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             join(6081..6149,6150..6941)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 6081 .. 6149 / #ccffcc / signal sequence
                        2: 6150 .. 6941 / #ccffcc"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
       61 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
      121 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
      181 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
      241 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
      301 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
      361 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
      421 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
      481 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
      541 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      601 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
      661 gccttttgct cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg
      721 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
      781 cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcacgc gtaacatatc
      841 ctggtgtgga gtaggggacg ctgctctgac agaggctcgg gggcctgagc tggctctgtg
      901 agctggggag gaggcagaca gccaggcctt gtctgcaagc agacctggca gcattgggct
      961 ggccgccccc cagggcctcc tcttcatgcc cagtgaatga ctcaccttgg cacagacaca
     1021 atgttcgggg tgggcacagt gcctgcttcc cgccgcaccc cagcccccct caaatgcctt
     1081 ccgagaagcc cattgagcag ggggcttgca ttgcacccca gcctgacagc ctggcatctt
     1141 gggataaaag cagcacagcc ccctaggggc tgcccttgct gtgtggcgcc accggcggtg
     1201 gagaacaagg ctctattcag cctgtgccca ggaaagggga tcaggggatg cccaggcatg
     1261 gacagtgggt ggcagggggg gagaggaggg ctgtctgctt cccagaagtc caaggacaca
     1321 aatgggtgag gggagagctc tccccatagc tgggctgcgg cccaacccca ccccctcagg
     1381 ctatgccagg gggtgttgcc aggggcaccc gggcatcgcc agtctagccc actccttcac
     1441 aaagccctcg catcccagga gcgagcagag ccagagcagg ttggagagga gacgcatcac
     1501 ctccgctgct cgctctagag ccgatccccg ggtaccgggc tagcgccacc atgggagact
     1561 acaaggatga cgacgataag ccaccggtca tgaagatgga caaaaagacc atcgtctggt
     1621 ttcggagaga tttgagaata gaagataatc ccgcgctcgc cgccgcggcc cacgagggtt
     1681 ccgtcttccc cgttttcatt tggtgtcctg aagaagaagg ccagttttat cccggaaggg
     1741 cctctaggtg gtggatgaag caaagtctgg cccatcttag ccagtcactg aaagcactgg
     1801 gcagtgatct taccctgatc aagacacaca ataccatctc tgccatcctc gactgcatca
     1861 gggtgaccgg cgcaacgaaa gtcgtgttta accacctgta cgatccagtt agtctggtgc
     1921 gcgaccacac tgtgaaggag aagctggtgg aacgggggat cagtgtgcag agctacaacg
     1981 gggaccttct gtacgagcca tgggagatct attgcgagaa agggaaaccg ttcacctcct
     2041 tcaacagtta ctggaagaaa tgtttggata tgtcaataga gtccgttatg ttgccccctc
     2101 cctggagact gatgccgatt actgctgctg cagaggccat ctgggcctgc tccatcgagg
     2161 aactcggtct ggaaaatgaa gcagaaaagc caagcaatgc acttctcact agagcctgga
     2221 gccccggctg gtctaatgcc gacaagctgc ttaacgagtt catcgaaaaa caactgattg
     2281 actacgcgaa gaactccaag aaagtggtag gtaactcaac tagcttgctc tctccatatc
     2341 tccattttgg cgagatttct gtccgccatg tatttcagtg cgctcggatg aaacagatta
     2401 tctgggctcg cgataaaaac agcgaaggcg ccgaaagcgc cgatctgttc ctgcgaggga
     2461 tcggacttcg ggaatactcc cggtatatat gtttcaactt tccattcaca cacgagcaga
     2521 gtctgttgtc ccacctcagg ttcttcccct gggacgccga tgtcgacaaa ttcaaggcat
     2581 ggagacaggg aaggacaggg tacccactcg tggatgctgg catgagagag ctctgggcta
     2641 caggctggat gcacaaccgc atccgggtaa tcgtgtcctc atttgctgtc aagtttctgc
     2701 tcctgccttg gaaatgggga atgaagtact tttgggatac ccttctcgac gccgacttgg
     2761 agtgtgacat tctgggatgg caatatatta gcgggtcaat tcctgacggc catgagttgg
     2821 acaggttgga caatccggcc ttgcagggag ctaagtatga tcccgaagga gagtatattc
     2881 gacagtggct ccccgagctg gcccgacttc ctacggagtg gattcaccat ccttgggacg
     2941 caccactgac agtgctcaag gcaagcgggg tggagctggg caccaattac gctaagccta
     3001 tagttgatat agatacagca cgcgagctgc tggctaaagc gatctctcgc actcgggagg
     3061 cgcagattat gatcggtgct gccgccaggg acccccccga cctcgacaac gaattcagtg
     3121 ggggaggcgg agggggtggt tcaggtggtg gtggtggtgg tggttcgggt ggtggcggag
     3181 ggggaggctc cggactcaga tctaaggagc acatgaagaa gatgatgaag gacttggagg
     3241 ggttacaccg agctgagcag agtctgcatg accttcagga aaggctgcac aaggcccagg
     3301 aggagcaccg cacagtggag gtggagaagg tccatctgga aaagaagctg cgcgatgaga
     3361 tcaaccttgc taagcaggaa gcccagcggc tgaaggagct gcgggagggt actgagaatg
     3421 agcggagccg ccaaaaatat gctgaggagg agttggagca ggttcgggag gccttgagga
     3481 aagcagagaa ggagctagaa tctcacagct catggtatgc tccagaggcc cttcagaagt
     3541 ggctgcagct gacacatgag gtggaggtgc aatattacaa catcaagaag caaaatgctg
     3601 agaagcagct gctggtggcc aaggaggggg ctgagaagat aaaaaagaag agaaacacac
     3661 tctttggcac cttccacgtg gcccacagct cttccctgga tgatgtagat cataaaattc
     3721 taacagctaa gcaagcactg agcgaggtga cagcagcatt gcgggagcgc ctgcaccgct
     3781 ggcaacagat cgagatcctc tgtggcttcc agattgtcaa caaccctggc atccactcac
     3841 tggtggctgc cctcaacata gaccccagct ggatgggcag tacacgcccc aaccctgctc
     3901 acttcatcat gactgacgac gtggatgaca tggatgagga gattgtgtct cccttgtcca
     3961 tgcagtcccc tagcctgcag agcagtgttc ggcagcgcct gacggagcca cagcatggcc
     4021 tgggatctca gagggatttg acccattccg attcggagtc ctccctccac atgagtgacc
     4081 gccagcgtgt ggcccccaaa cctcctcaga tgagccgtgc tgcagacgag gctctcaatg
     4141 ccatgacttc caatggcagc caccggctga tcgagggggt ccacccaggg tctctggtgg
     4201 agaaactgcc tgacagccct gccctggcca agaaggcatt actggcgctg aaccatgggc
     4261 tggacaaggc ccacagcctg atggagctga gcccctcagc cccacctggt ggctctccac
     4321 atttggattc ttcccgttct cacagcccca gctccccaga cccagacaca ccatctccag
     4381 ttggggacag ccgagccctg caagccagcc gaaacacacg cattccccac ctggctggca
     4441 agaaggctgt ggctgaggag gataatggct ctattggcga ggaaacagac tccagcccag
     4501 gccggaagaa gtttcctctc aaaatcttta agaagcctct taagaagtag gaattgggat
     4561 ccctcgagat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     4621 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     4681 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttag ttcttgccac
     4741 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     4801 tgacaattcc gtggtgttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4861 ctagctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     4921 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt
     4981 gggaggtttt ttaaagcggc cgcaggacgg accgagcggc cgcaggaacc cctagtgatg
     5041 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     5101 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     5161 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5221 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5281 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5341 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5401 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     5461 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5521 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5581 gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5641 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     5701 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5761 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5821 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5881 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5941 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6001 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6061 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6121 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6181 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6241 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6301 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6361 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6421 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6481 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6541 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6601 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6661 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6721 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6781 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6841 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6901 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6961 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     7021 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7081 caga
//
