LOCUS       sequence-450141-        6296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of FLAG-tagged-TurboID and T2A-mCherry under the GFAP 
            short promoter by AAV vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6296)
  AUTHORS   Sun X, Sun H, Han X, Chen PC, Jiao Y, Wu Z, Zhang X, Wang Z, Niu M, 
            Yu K, Liu D, Dey KK, Mancieri A, Fu Y, Cho JH, Li Y, Poudel S, 
            Branon TC, Ting AY, Peng J
  TITLE     Deep Single-Cell-Type Proteome Profiling of Mouse Brain by 
            Nonsurgical AAV-Mediated Proximity Labeling.
  JOURNAL   Anal Chem. 2022 Apr 5;94(13):5325-5334. doi: 
            10.1021/acs.analchem.1c05212. Epub 2022 Mar 22.
  PUBMED    35315655
REFERENCE   2  (bases 1 to 6296)
  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"; doi: 
            "10.1021/acs.analchem.1c05212"; journalName: "Anal Chem"; date: 
            "2022-04-5- 5"; volume: "94"; issue: "13"; pages: "5325-5334"
COMMENT     Sequence Label: AAV-GFAP-TurboID
FEATURES             Location/Qualifiers
     source          1..6296
                     /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
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    849..873
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      874..883
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             883..948
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             955..1911
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             1921..1974
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1975..2685
                     /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(2120..2138)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2388..2407
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2686..2710)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2720..3308
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2773..2793)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3191..3202)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3338..3355)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3344..3551
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3559..3699
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3559..3688
                     /label=AAV2 ITR
     rep_origin      3774..4229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3861..3880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4071..4092
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4246..4265)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4365..4387
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4425..4443)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4511..4615
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4616..4684,4685..5476)
                     /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"
     rep_origin      5647..6235
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6136..6155
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgaa catatcctgg
      181 tgtggagtag gggacgctgc tctgacagag gctcgggggc ctgagctggc tctgtgagct
      241 ggggaggagg cagacagcca ggccttgtct gcaagcagac ctggcagcat tgggctggcc
      301 gccccccagg gcctcctctt catgcccagt gaatgactca ccttggcaca gacacaatgt
      361 tcggggtggg cacagtgcct gcttcccgcc gcaccccagc ccccctcaaa tgccttccga
      421 gaagcccatt gagcaggggg cttgcattgc accccagcct gacagcctgg catcttggga
      481 taaaagcagc acagccccct aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga
      541 acaaggctct attcagcctg tgcccaggaa aggggatcag gggatgccca ggcatggaca
      601 gtgggtggca gggggggaga ggagggctgt ctgcttccca gaagtccaag gacacaaatg
      661 ggtgagggga gagctctccc catagctggg ctgcggccca accccacccc ctcaggctat
      721 gccagggggt gttgccaggg gcacccgggc atcgccagtc tagcccactc cttcataaag
      781 ccctcgcatc ccaggagcga gcagagccag agcaggttgg agaggagacg catcacctcc
      841 gctgctcgcc aagtttgtac aaaaaagcag gctgccacca tggactacaa agaccatgac
      901 ggtgattata aagatcatga tatcgattac aaggatgacg atgacaaggc tagcaaagac
      961 aatactgtgc ctctgaagct gatcgctctc ctggctaatg gcgagttcca tagtggcgaa
     1021 cagctgggag aaaccctggg catgtccagg gccgctatca acaagcacat tcagactctg
     1081 cgcgactggg gcgtggacgt gttcaccgtg cccggaaagg gctactctct gcccgagcct
     1141 atcccgctgc tgaacgctaa acagattctg ggacagctgg acggcgggag cgtggcagtc
     1201 ctgcctgtgg tcgactccac caatcagtac ctgctggatc gaatcggcga gctgaagagt
     1261 ggggatgctt gcattgcaga atatcagcag gcagggagag gaagcagagg gaggaaatgg
     1321 ttctctcctt ttggagctaa cctgtacctg agtatgtttt ggcgcctgaa gcggggacca
     1381 gcagcaatcg gcctgggccc ggtcatcgga attgtcatgg cagaagcgct gcgaaagctg
     1441 ggagcagaca aggtgcgagt caaatggccc aatgacctgt atctgcagga tagaaagctg
     1501 gcaggcatcc tggtggagct ggccggaata acaggcgatg ctgcacagat cgtcattggc
     1561 gccgggatta acgtggctat gaggcgcgtg gaggaaagcg tggtcaatca gggctggatc
     1621 acactgcagg aagcagggat taacctggac aggaatactc tggccgctac gctgatccga
     1681 gagctgcggg cagccctgga actgttcgag caggaaggcc tggctccata tctgccacgg
     1741 tgggagaagc tggataactt catcaataga cccgtgaagc tgatcattgg ggacaaagag
     1801 attttcggga ttagccgggg gattgataaa cagggagccc tgctgctgga acaggacgga
     1861 gttatcaaac cctggatggg cggagaaatc agtctgcggt ctgccgaaaa gggaagcgga
     1921 gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg ccccatggtg
     1981 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     2041 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     2101 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     2161 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     2221 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     2281 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     2341 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     2401 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     2461 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     2521 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     2581 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     2641 gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaaccca gctttcttgt
     2701 acaaagtggg aattccgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     2761 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     2821 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     2881 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     2941 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3001 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3061 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac
     3121 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     3181 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     3241 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     3301 ctccccgcat cgggaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     3361 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3421 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3481 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg
     3541 catgctgggg agggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     3601 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     3661 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     3721 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     3781 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3841 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3901 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3961 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     4021 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4081 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     4141 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4201 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     4261 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4321 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4381 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     4441 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     4501 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4561 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4621 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4681 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4741 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4801 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4861 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4921 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4981 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5041 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5101 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5161 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5221 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5281 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     5341 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5401 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5461 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5521 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5581 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5641 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5701 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5761 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5821 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5881 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5941 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6001 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6061 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6121 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6181 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6241 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
