LOCUS       sequence-348675-        6921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses membrane bound biotin ligase, BioID2, in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6921)
  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 6921)
  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-Lck-13xLinker-BioID2-BioID2-HA
FEATURES             Location/Qualifiers
     source          1..6921
                     /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"
     CDS             1752..2450
                     /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             2535..3233
                     /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             3243..3269
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3294..3882
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3347..3367)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3765..3776)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3946..4067
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3957..3976)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4011..4030
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4229..4358
                     /label=AAV2 ITR
     primer_bind     complement(4377..4394)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4377..4393)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4386..4408)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4535..4990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4622..4641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4832..4853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5016..5120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5121..5189,5190..5981)
                     /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      6152..6740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6641..6660
                     /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 tcgagctcgg atccaccacc atgggctgtg gctgcagctc aaaccctgaa gatgactgga
     1501 tggagaacat tgacgtgtgt gagaactgcc attatccccc gccgggtgga ggcgggtctg
     1561 gaggcggggg tagtggcggg ggtggaagcg ggggtggagg cgggtcgggt ggcggaggta
     1621 gcggaggcgg tggaagtgga ggcggtggaa gtggtggagg aggttctggt ggcggaggta
     1681 gcggtggcgg aggtagcgga ggcggtggaa gtggtggcgg aggtagcgga ggcggtggat
     1741 cggtcgacac gatgttcaaa aatcttattt ggctgaagga agttgatagc acccaggagc
     1801 ggcttaagga atggaatgtc tcctatggca ctgcactcgt cgccgataga cagaccaaag
     1861 gtagaggcgg tctcggacgg aagtggttga gtcaggaggg cggtctttac ttctctttct
     1921 tgttgaatcc aaaagagttt gaaaatttgt tgcaacttcc actggtcttg ggactctccg
     1981 tgagcgaggc actcgaagag attacagaga taccatttag tctgaagtgg cccaacgacg
     2041 tttattttca ggagaaaaag gtttcagggg tcctttgtga gctcagtaag gacaagctga
     2101 tagtgggaat cggtataaac gtgaatcaaa gagagatccc tgaagagatc aaggatcggg
     2161 ccacaacact ttatgaaatc actggaaagg attgggacag aaaggaggtc ttgctgaagg
     2221 ttctcaagcg aatttccgaa aatctcaaga aatttaagga aaaatccttt aaagagttca
     2281 aaggaaaaat tgagtcaaag atgttgtacc ttggagagga ggttaaactc ttgggagaag
     2341 gcaagattac cgggaaactt gtgggcctga gtgaaaaggg cggtgctttg atacttacag
     2401 aggaaggcat aaaggagata ctgtccggcg aatttagctt gcgacggtca agcttaggtg
     2461 gaggaggttc tggaggcggt ggaagtggtg gcggaggtag cggtggagga ggttctatgg
     2521 tgtctaagga attcatgttc aaaaatctta tttggctgaa ggaagttgat agcacccagg
     2581 agcggcttaa ggaatggaat gtctcctatg gcactgcact cgtcgccgat agacagacca
     2641 aaggtagagg cggtctcgga cggaagtggt tgagtcagga gggcggtctt tacttctctt
     2701 tcttgttgaa tccaaaagag tttgaaaatt tgttgcaact tccactggtc ttgggactct
     2761 ccgtgagcga ggcactcgaa gagattacag agataccatt tagtctgaag tggcccaacg
     2821 acgtttattt tcaggagaaa aaggtttcag gggtcctttg tgagctcagt aaggacaagc
     2881 tgatagtggg aatcggtata aacgtgaatc aaagagagat ccctgaagag atcaaggatc
     2941 gggccacaac actttatgaa atcactggaa aggattggga cagaaaggag gtcttgctga
     3001 aggttctcaa gcgaatttcc gaaaatctca agaaatttaa ggaaaaatcc tttaaagagt
     3061 tcaaaggaaa aattgagtca aagatgttgt accttggaga ggaggttaaa ctcttgggag
     3121 aaggcaagat taccgggaaa cttgtgggcc tgagtgaaaa gggcggtgct ttgatactta
     3181 cagaggaagg cataaaggag atactgtccg gcgaatttag cttgcgacgg tcagctagcg
     3241 cttaccctta cgatgtaccg gattacgcat agggatccaa ttcgcttatc gataatcaac
     3301 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     3361 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     3421 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     3481 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     3541 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     3601 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     3661 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg
     3721 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     3781 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     3841 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cggaattctg
     3901 cagatatcta gagtcgaccc gggcggccgc ttcgagcaga catgataaga tacattgatg
     3961 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4021 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4081 gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa agcaagtaaa
     4141 acctctacaa atgtggtaaa atcgataagg atcttcctag agcatggcta cgtagataag
     4201 tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc
     4261 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4321 tttgcccggg cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc taattcactg
     4381 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     4441 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     4501 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     4561 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     4621 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4681 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4741 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4801 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4861 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4921 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4981 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5041 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5101 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5161 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5221 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5281 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5341 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5401 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5461 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5521 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5581 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5641 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5701 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5761 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5821 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5881 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5941 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6001 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6061 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6121 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6181 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6241 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6301 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6361 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6421 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6481 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6541 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6601 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6661 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6721 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6781 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6841 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6901 cagtgagcga ggaagcggaa g
//
