LOCUS       sequence-368422-        7540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Aquaporin-4 fused with BioID2 at the C-terminus in 
            astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7540)
  AUTHORS   Soto JS, Jami-Alahmadi Y, Chacon J, Moye SL, Diaz-Castro B, 
            Wohlschlegel JA, Khakh BS
  TITLE     Astrocyte-neuron subproteomes and obsessive-compulsive disorder 
            mechanisms.
  JOURNAL   Nature. 2023 Apr;616(7958):764-773. doi: 10.1038/s41586-023-05927-7.
            Epub 2023 Apr 12.
  PUBMED    37046092
REFERENCE   2  (bases 1 to 7540)
  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: 
            "10.1038/s41586-023-05927-7"; journalName: "Nature"; date: 
            "2023-04"; volume: "616"; issue: "7958"; pages: "764-773"
COMMENT     Sequence Label: pZac2.1 gfaABC1D-Aqp4-BioID2-BioID2-HA
FEATURES             Location/Qualifiers
     source          1..7540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     intron          1239..1371
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1354..1373
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1416..1434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1445..1454
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2371..3069
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKW
                     LSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKV
                     SGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISE
                     NLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKE
                     ILSGEFSLRRS"
     CDS             3154..3852
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKW
                     LSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKV
                     SGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISE
                     NLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKE
                     ILSGEFSLRRS"
     CDS             3862..3888
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3913..4501
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3966..3986)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4384..4395)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4565..4686
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4576..4595)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4630..4649
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4848..4977
                     /label=AAV2 ITR
     primer_bind     complement(4996..5013)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4996..5012)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5005..5027)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5154..5609
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5241..5260)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5451..5472
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5635..5739
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5740..5808,5809..6600)
                     /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      6771..7359
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7260..7279
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag gggacgctgc
      481 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      541 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      601 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      661 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      721 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      781 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      841 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      901 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      961 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
     1021 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
     1081 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca agctttattg
     1141 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     1201 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     1261 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     1321 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1381 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     1441 tcgaccatgg tggctttcaa aggagtctgg actcaggctt tctggaaggc agtctcagca
     1501 gaatttctgg ccacgcttat ctttgttttg ctcggtgtgg gatccaccat aaactggggt
     1561 ggctcagaaa accccttacc tgtggacatg gtcctcatct ccctttgctt tggactcagc
     1621 attgctacca tggtgcagtg ctttggccac atcagtggtg gccacatcaa tcccgctgtg
     1681 actgtagcca tggtgtgcac acgaaagatc agcatcgcta agtccgtctt ctacatcatt
     1741 gcacagtgcc tgggggccat cattggagcc ggcatcctct acctggtcac acctcccagt
     1801 gtggttggag gattgggagt caccacggtt catggaaacc tcaccgctgg ccatgggctc
     1861 ctggtggagt taataatcac tttccagttg gtgttcacta tttttgccag ctgtgattcc
     1921 aaacgaactg atgttactgg ttcaatagct ttagcaattg gattttccgt tgcaattgga
     1981 catttgtttg caatcaatta tactggagcc agcatgaatc cagctcgatc ttttggaccc
     2041 gcagttatca tgggaaactg ggcaaaccac tggatatatt gggttggacc aatcatgggc
     2101 gctgtgctgg caggtgccct ttatgagtat gtcttctgtc ctgatgtgga gctcaaacgt
     2161 cgccttaagg aagccttcag caaagccgcg cagcagacaa aagggagcta catggaggtg
     2221 gaggacaacc ggagccaagt ggagacggaa gacttgatcc tgaagcccgg agtggtgcat
     2281 gtgattgaca ttgaccgtgg agaagagaag aaggggaaag actcttcggg agaggtattg
     2341 tcttccgtac tcgagctcgg atccaccacc atgttcaaaa atcttatttg gctgaaggaa
     2401 gttgatagca cccaggagcg gcttaaggaa tggaatgtct cctatggcac tgcactcgtc
     2461 gccgatagac agaccaaagg tagaggcggt ctcggacgga agtggttgag tcaggagggc
     2521 ggtctttact tctctttctt gttgaatcca aaagagtttg aaaatttgtt gcaacttcca
     2581 ctggtcttgg gactctccgt gagcgaggca ctcgaagaga ttacagagat accatttagt
     2641 ctgaagtggc ccaacgacgt ttattttcag gagaaaaagg tttcaggggt cctttgtgag
     2701 ctcagtaagg acaagctgat agtgggaatc ggtataaacg tgaatcaaag agagatccct
     2761 gaagagatca aggatcgggc cacaacactt tatgaaatca ctggaaagga ttgggacaga
     2821 aaggaggtct tgctgaaggt tctcaagcga atttccgaaa atctcaagaa atttaaggaa
     2881 aaatccttta aagagttcaa aggaaaaatt gagtcaaaga tgttgtacct tggagaggag
     2941 gttaaactct tgggagaagg caagattacc gggaaacttg tgggcctgag tgaaaagggc
     3001 ggtgctttga tacttacaga ggaaggcata aaggagatac tgtccggcga atttagcttg
     3061 cgacggtcaa gcttaggtgg aggaggttct ggaggcggtg gaagtggtgg cggaggtagc
     3121 ggtggaggag gttctatggt gtctaaggaa ttcatgttca aaaatcttat ttggctgaag
     3181 gaagttgata gcacccagga gcggcttaag gaatggaatg tctcctatgg cactgcactc
     3241 gtcgccgata gacagaccaa aggtagaggc ggtctcggac ggaagtggtt gagtcaggag
     3301 ggcggtcttt acttctcttt cttgttgaat ccaaaagagt ttgaaaattt gttgcaactt
     3361 ccactggtct tgggactctc cgtgagcgag gcactcgaag agattacaga gataccattt
     3421 agtctgaagt ggcccaacga cgtttatttt caggagaaaa aggtttcagg ggtcctttgt
     3481 gagctcagta aggacaagct gatagtggga atcggtataa acgtgaatca aagagagatc
     3541 cctgaagaga tcaaggatcg ggccacaaca ctttatgaaa tcactggaaa ggattgggac
     3601 agaaaggagg tcttgctgaa ggttctcaag cgaatttccg aaaatctcaa gaaatttaag
     3661 gaaaaatcct ttaaagagtt caaaggaaaa attgagtcaa agatgttgta ccttggagag
     3721 gaggttaaac tcttgggaga aggcaagatt accgggaaac ttgtgggcct gagtgaaaag
     3781 ggcggtgctt tgatacttac agaggaaggc ataaaggaga tactgtccgg cgaatttagc
     3841 ttgcgacggt cagctagcgc ttacccttac gatgtaccgg attacgcata gggatccaat
     3901 tcgcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3961 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     4021 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     4081 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     4141 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     4201 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     4261 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     4321 ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     4381 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     4441 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     4501 catcgatacc ggaattctgc agatatctag agtcgacccg ggcggccgct tcgagcagac
     4561 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     4621 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     4681 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag
     4741 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tcttcctaga
     4801 gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt
     4861 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     4921 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct
     4981 taattaacct aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     5041 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     5101 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     5161 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     5221 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     5281 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     5341 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     5401 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5461 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     5521 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5581 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     5641 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5701 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5761 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5821 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5881 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5941 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6001 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6061 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6121 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6181 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6241 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6301 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6361 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6421 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6481 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6541 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6601 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6661 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6721 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6781 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6841 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6901 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6961 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7021 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7081 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7141 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7201 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7261 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7321 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7381 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     7441 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     7501 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
//
