LOCUS       sequence-450139-        6952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of FLAG-tagged TurboID and T2A-mCherry under the 
            CaMKII-alpha short promoter by AAV vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6952)
  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 6952)
  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-CaMKII-TurboID
FEATURES             Location/Qualifiers
     source          1..6952
                     /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"
     promoter        180..1468
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     primer_bind     complement(1481..1497)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1481..1497)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    1505..1529
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1530..1539
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1539..1604
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1611..2567
                     /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             2577..2630
                     /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             2631..3341
                     /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(2776..2794)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3044..3063
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(3342..3366)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3376..3964
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3429..3449)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3847..3858)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3994..4011)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4000..4207
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4215..4355
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4215..4344
                     /label=AAV2 ITR
     rep_origin      4430..4885
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4517..4536)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4727..4748
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4902..4921)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5021..5043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5081..5099)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5167..5271
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5272..5340,5341..6132)
                     /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      6303..6891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6792..6811
                     /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 aaaagttgcc cttaattaac
      181 attatggcct taggtcactt catctccatg gggttcttct tctgattttc tagaaaatga
      241 gatgggggtg cagagagctt cctcagtgac ctgcccaggg tcacatcaga aatgtcagag
      301 ctagaacttg aactcagatt actaatctta aattccatgc cttgggggca tgcaagtacg
      361 atatacagaa ggagtgaact cattagggca gatgaccaat gagtttagga aagaagagtc
      421 cagggcaggg tacatctaca ccacccgccc agccctgggt gagtccagcc acgttcacct
      481 cattatagtt gcctctctcc agtcctacct tgacgggaag cacaagcaga aactgggaca
      541 ggagccccag gagaccaaat cttcatggtc cctctgggag gatgggtggg gagagctgtg
      601 gcagaggcct caggaggggc cctgctgctc agtggtgaca gataggggtg agaaagcaga
      661 cagagtcatt ccgtcagcat tctgggtctg tttggtactt cttctcacgc taaggtggcg
      721 gtgtgatatg cacaatggct aaaaagcagg gagagctgga aagaaacaag gacagagaca
      781 gaggccaagt caaccagacc aattcccaga ggaagcaaag aaaccattac agagactaca
      841 agggggaagg gaaggagaga tgaattagct tcccctgtaa accttagaac ccagctgttg
      901 ccagggcaac ggggcaatac ctgtctcttc agaggagatg aagttgccag ggtaactaca
      961 tcctgtcttt ctcaaggacc atcccagaat gtggcaccca ctagccgtta ccatagcaac
     1021 tgcctctttg ccccacttaa tcccatcccg tctgttaaaa gggccctata gttggaggtg
     1081 ggggaggtag gaagagcgat gatcacttgt ggactaagtt tgttcgcatc cccttctcca
     1141 accccctcag tacatcaccc tgggggaaca gggtccactt gctcctgggc ccacacagtc
     1201 ctgcagtatt gtgtatataa ggccagggca aagaggagca ggttttaaag tgaaaggcag
     1261 gcaggtgttg gggaggcagt taccggggca acgggaacag ggcgtttcgg aggtggttgc
     1321 catggggacc tggatgctga cgaaggctcg cgaggctgtg agcagccaca gtgccctgct
     1381 cagaagcccc aagctcgtca gtcaagccgg ttctccgttt gcactcagga gcacgggcag
     1441 gcgagtggcc cctagttctg ggggcagctc tagagcgggg gatccactag ttctagagcg
     1501 gccgccaagt ttgtacaaaa aagcaggctg ccaccatgga ctacaaagac catgacggtg
     1561 attataaaga tcatgatatc gattacaagg atgacgatga caaggctagc aaagacaata
     1621 ctgtgcctct gaagctgatc gctctcctgg ctaatggcga gttccatagt ggcgaacagc
     1681 tgggagaaac cctgggcatg tccagggccg ctatcaacaa gcacattcag actctgcgcg
     1741 actggggcgt ggacgtgttc accgtgcccg gaaagggcta ctctctgccc gagcctatcc
     1801 cgctgctgaa cgctaaacag attctgggac agctggacgg cgggagcgtg gcagtcctgc
     1861 ctgtggtcga ctccaccaat cagtacctgc tggatcgaat cggcgagctg aagagtgggg
     1921 atgcttgcat tgcagaatat cagcaggcag ggagaggaag cagagggagg aaatggttct
     1981 ctccttttgg agctaacctg tacctgagta tgttttggcg cctgaagcgg ggaccagcag
     2041 caatcggcct gggcccggtc atcggaattg tcatggcaga agcgctgcga aagctgggag
     2101 cagacaaggt gcgagtcaaa tggcccaatg acctgtatct gcaggataga aagctggcag
     2161 gcatcctggt ggagctggcc ggaataacag gcgatgctgc acagatcgtc attggcgccg
     2221 ggattaacgt ggctatgagg cgcgtggagg aaagcgtggt caatcagggc tggatcacac
     2281 tgcaggaagc agggattaac ctggacagga atactctggc cgctacgctg atccgagagc
     2341 tgcgggcagc cctggaactg ttcgagcagg aaggcctggc tccatatctg ccacggtggg
     2401 agaagctgga taacttcatc aatagacccg tgaagctgat cattggggac aaagagattt
     2461 tcgggattag ccgggggatt gataaacagg gagccctgct gctggaacag gacggagtta
     2521 tcaaaccctg gatgggcgga gaaatcagtc tgcggtctgc cgaaaaggga agcggagagg
     2581 gcaggggaag tcttctaaca tgcggggacg tggaggaaaa tcccggcccc atggtgagca
     2641 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     2701 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     2761 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     2821 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     2881 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     2941 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     3001 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     3061 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     3121 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     3181 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     3241 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     3301 gccgccactc caccggcggc atggacgagc tgtacaagta aacccagctt tcttgtacaa
     3361 agtgggaatt ccgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3421 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3481 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3541 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3601 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3661 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3721 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc
     3781 tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3841 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3901 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     3961 ccgcatcggg aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     4021 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     4081 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4141 gggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
     4201 ctggggaggg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4261 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4321 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4381 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4441 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4501 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4561 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4621 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4681 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4741 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     4801 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4861 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4921 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4981 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5041 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     5101 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     5161 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5221 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5281 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5341 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5401 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5461 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5521 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5581 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5641 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5701 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5761 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5821 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5881 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5941 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     6001 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6061 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6121 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6181 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6241 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6301 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6361 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6421 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6481 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6541 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6601 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6661 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6721 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6781 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6841 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6901 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
