LOCUS       sequence-439323-        5560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Secreted HA-tagged TurboID for extracellular proximity labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5560)
  AUTHORS   Irala D, Wang S, Sakers K, Nagendren L, Ulloa Severino FP, Bindu DS,
            Savage JT, Eroglu C
  TITLE     Astrocyte-secreted neurocan controls inhibitory synapse formation 
            and function.
  JOURNAL   Neuron. 2024 Mar 27:S0896-6273(24)00161-2. doi: 
            10.1016/j.neuron.2024.03.007.
  PUBMED    38574730
REFERENCE   2  (bases 1 to 5560)
  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"; journalName: "Neuron. 
            2024 Mar 27:S0896-6273(24)00161-2. doi: 
            10.1016/j.neuron.2024.03.007."
COMMENT     Sequence Label: pZac2.1-GfaABC1D -IgK-TurboID-HA
FEATURES             Location/Qualifiers
     source          1..5560
                     /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      1469..1478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     1475..1534
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             1556..2515
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             2519..2545
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2585..2706
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2596..2615)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2650..2669
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2868..2997
                     /label=AAV2 ITR
     primer_bind     complement(3016..3033)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3016..3032)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3025..3047)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3174..3629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3261..3280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3471..3492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3655..3759
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3760..3828,3829..4620)
                     /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      4791..5379
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5280..5299
                     /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 tcgagaattc gcggcccagg cggcccaggc caccatggag acagacacac tcctgctatg
     1501 ggtactgctg ctctgggttc caggttccac tggtgacgcg gcccaggaat tcaccatgaa
     1561 agacaatact gtgcctctga agctgatcgc tctcctggct aatggcgagt tccatagtgg
     1621 cgaacagctg ggagaaaccc tgggcatgtc cagggccgct atcaacaagc acattcagac
     1681 tctgcgcgac tggggcgtgg acgtgttcac cgtgcccgga aagggctact ctctgcccga
     1741 gcctatcccg ctgctgaacg ctaaacagat tctgggacag ctggacggcg ggagcgtggc
     1801 agtcctgcct gtggtcgact ccaccaatca gtacctgctg gatcgaatcg gcgagctgaa
     1861 gagtggggat gcttgcattg cagaatatca gcaggcaggg agaggaagca gagggaggaa
     1921 atggttctct ccttttggag ctaacctgta cctgagtatg ttttggcgcc tgaagcgggg
     1981 accagcagca atcggcctgg gcccggtcat cggaattgtc atggcagaag cgctgcgaaa
     2041 gctgggagca gacaaggtgc gagtcaaatg gcccaatgac ctgtatctgc aggatagaaa
     2101 gctggcaggc atcctggtgg agctggccgg aataacaggc gatgctgcac agatcgtcat
     2161 tggcgccggg attaacgtgg ctatgaggcg cgtggaggaa agcgtggtca atcagggctg
     2221 gatcacactg caggaagcag ggattaacct ggacaggaat actctggccg ctacgctgat
     2281 ccgagagctg cgggcagccc tggaactgtt cgagcaggaa ggcctggctc catatctgcc
     2341 acggtgggag aagctggata acttcatcaa tagacccgtg aagctgatca ttggggacaa
     2401 agagattttc gggattagcc gggggattga taaacaggga gccctgctgc tggaacagga
     2461 cggagttatc aaaccctgga tgggcggaga aatcagtctg cggtctgccg aaaagcttta
     2521 cccttacgat gtaccggatt acgcatctag agtcgacccg ggcggccgct tcgagcagac
     2581 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     2641 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     2701 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag
     2761 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tcttcctaga
     2821 gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt
     2881 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     2941 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct
     3001 taattaacct aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     3061 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     3121 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     3181 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3241 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3301 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3361 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3421 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3481 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3541 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3601 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     3661 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3721 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3781 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3841 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3901 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3961 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     4021 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     4081 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4141 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4201 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4261 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     4321 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4381 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4441 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4501 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4561 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4621 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4681 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4741 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4801 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4861 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4921 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4981 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5041 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5101 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5161 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5221 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5281 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5341 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5401 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5461 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5521 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
//
