LOCUS       sequence-348693-        7787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Connexin-43 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 7787)
  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 7787)
  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-Cx43-BioID2-BioID2-HA
FEATURES             Location/Qualifiers
     source          1..7787
                     /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             2618..3316
                     /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             3401..4099
                     /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             4109..4135
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    4160..4748
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4213..4233)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4631..4642)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4812..4933
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4823..4842)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4877..4896
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5095..5224
                     /label=AAV2 ITR
     primer_bind     complement(5243..5260)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5243..5259)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5252..5274)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5401..5856
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5488..5507)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5698..5719
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5882..5986
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5987..6055,6056..6847)
                     /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      7018..7606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7507..7526
                     /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 tcgagccacc atgggtgact ggagtgcctt ggggaagctt ctggacaagg tccaagccta
     1501 ctccaccgct ggagggaagg tgtggctgtc agtgctcttc atattcagaa tcctgctcct
     1561 ggggacagct gttgagtcag cttggggtga tgaacagtct gcctttcgct gtaacactca
     1621 acaacctggc tgcgaaaacg tctgctatga caagtccttc cccatctctc acgtgcgctt
     1681 ctgggtcctt cagatcatat tcgtgtctgt gcccaccctc ctgtacttgg cccatgtgtt
     1741 ctatgtgatg aggaaggaag agaagctaaa caagaaagaa gaggagctca aagtggccca
     1801 gactgacggg gtcaacgtgg agatgcacct gaagcagatt gaaatcaaga agttcaagta
     1861 cgggattgaa gagcacggca aggtgaaaat gaggggcggc ttgctgagaa cctacatcat
     1921 cagcatcctc ttcaagtctg tcttcgaggt ggccttcctg ctcatccagt ggtacatcta
     1981 tgggttcagc ttgagcgcgg tctacacctg caagagagat ccctgccccc accaggtaga
     2041 ctgcttcctc tcacgtccca cggagaaaac catcttcatc atcttcatgc tggtggtgtc
     2101 cttggtgtct ctcgctttga acatcattga gctcttctac gtcttcttca aaggcgttaa
     2161 ggatcgcgtg aagggaagaa gcgatcctta ccacgccacc actggcccac tgagcccatc
     2221 aaaagactgc ggatctccaa aatacgccta cttcaatggc tgctcctcac caacggctcc
     2281 actctcgcct atgtctcctc ctgggtacaa gctggttact ggtgacagaa acaattcctc
     2341 gtgccgcaat tacaacaagc aagctagcga gcaaaactgg gcgaactaca gcgcagagca
     2401 aaatcgcatg gggcaggccg gaagcaccat ctccaactcg cacgcccagc cgttcgattt
     2461 ccccgacgac aaccagaatg ccaaaaaagt tgctgctgga catgaactcc agccattagc
     2521 catcgtggac caacgacctt ccagcagagc cagcagccgc gccagcagca ggcctcggcc
     2581 tgatgacctg gagattctcg agctcggatc caccaccatg ttcaaaaatc ttatttggct
     2641 gaaggaagtt gatagcaccc aggagcggct taaggaatgg aatgtctcct atggcactgc
     2701 actcgtcgcc gatagacaga ccaaaggtag aggcggtctc ggacggaagt ggttgagtca
     2761 ggagggcggt ctttacttct ctttcttgtt gaatccaaaa gagtttgaaa atttgttgca
     2821 acttccactg gtcttgggac tctccgtgag cgaggcactc gaagagatta cagagatacc
     2881 atttagtctg aagtggccca acgacgttta ttttcaggag aaaaaggttt caggggtcct
     2941 ttgtgagctc agtaaggaca agctgatagt gggaatcggt ataaacgtga atcaaagaga
     3001 gatccctgaa gagatcaagg atcgggccac aacactttat gaaatcactg gaaaggattg
     3061 ggacagaaag gaggtcttgc tgaaggttct caagcgaatt tccgaaaatc tcaagaaatt
     3121 taaggaaaaa tcctttaaag agttcaaagg aaaaattgag tcaaagatgt tgtaccttgg
     3181 agaggaggtt aaactcttgg gagaaggcaa gattaccggg aaacttgtgg gcctgagtga
     3241 aaagggcggt gctttgatac ttacagagga aggcataaag gagatactgt ccggcgaatt
     3301 tagcttgcga cggtcaagct taggtggagg aggttctgga ggcggtggaa gtggtggcgg
     3361 aggtagcggt ggaggaggtt ctatggtgtc taaggaattc atgttcaaaa atcttatttg
     3421 gctgaaggaa gttgatagca cccaggagcg gcttaaggaa tggaatgtct cctatggcac
     3481 tgcactcgtc gccgatagac agaccaaagg tagaggcggt ctcggacgga agtggttgag
     3541 tcaggagggc ggtctttact tctctttctt gttgaatcca aaagagtttg aaaatttgtt
     3601 gcaacttcca ctggtcttgg gactctccgt gagcgaggca ctcgaagaga ttacagagat
     3661 accatttagt ctgaagtggc ccaacgacgt ttattttcag gagaaaaagg tttcaggggt
     3721 cctttgtgag ctcagtaagg acaagctgat agtgggaatc ggtataaacg tgaatcaaag
     3781 agagatccct gaagagatca aggatcgggc cacaacactt tatgaaatca ctggaaagga
     3841 ttgggacaga aaggaggtct tgctgaaggt tctcaagcga atttccgaaa atctcaagaa
     3901 atttaaggaa aaatccttta aagagttcaa aggaaaaatt gagtcaaaga tgttgtacct
     3961 tggagaggag gttaaactct tgggagaagg caagattacc gggaaacttg tgggcctgag
     4021 tgaaaagggc ggtgctttga tacttacaga ggaaggcata aaggagatac tgtccggcga
     4081 atttagcttg cgacggtcag ctagcgctta cccttacgat gtaccggatt acgcataggg
     4141 atccaattcg cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     4201 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     4261 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     4321 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     4381 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     4441 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     4501 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     4561 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     4621 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4681 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4741 ctccccgcat cgataccgga attctgcaga tatctagagt cgacccgggc ggccgcttcg
     4801 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     4861 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     4921 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat
     4981 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatct
     5041 tcctagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac
     5101 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     5161 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     5221 gcagccttaa ttaacctaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     5281 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     5341 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     5401 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5461 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5521 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5581 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5641 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5701 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5761 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5821 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     5881 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5941 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     6001 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     6061 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     6121 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6181 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     6241 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6301 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6361 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6421 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6481 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6541 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6601 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6661 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6721 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6781 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6841 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6901 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6961 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     7021 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     7081 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     7141 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     7201 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     7261 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     7321 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7381 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7441 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7501 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7561 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7621 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     7681 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     7741 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaag
//
