LOCUS       sequence-439372-        6661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Secreted HA-tagged TurboID-NCAN-IL domain for extracellular 
            proximity labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6661)
  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 6661)
  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- NCAN IL-HA
FEATURES             Location/Qualifiers
     source          1..6661
                     /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"
     regulatory      2606..2615
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3635..3661
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3686..3807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3697..3716)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3751..3770
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3969..4098
                     /label=AAV2 ITR
     primer_bind     complement(4117..4134)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4117..4133)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4126..4148)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4275..4730
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4362..4381)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4572..4593
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4756..4860
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4861..4929,4930..5721)
                     /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      5892..6480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6381..6400
                     /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 atcggaattc ggctccaccg gctccaccgg
     2581 ctccaccggc gcggatccac cggtcgccac catggacacc accgccacgg aaaaggggct
     2641 tcgcatgctg aagtcagggt caggacccgt ccgtgctgcc ctggcagagc tagtggccct
     2701 gccctgcttc tttaccctgc aaccacggct aagctccctg cgagacattc ctcggatcaa
     2761 gtggactaag gttcagactg catcaggcca gcgacaggat ttgccaatct tggtggccaa
     2821 agacaacgtg gtgcgtgtgg ccaagggctg gcagggacgg gtgtcattgc ctgcttatcc
     2881 ccggcacaga gccaatgcta cccttctgct ggggccactt cgagcaagcg actctgggct
     2941 gtatcgctgc caagtggtaa agggtatcga agatgagcag gacctggtaa ccctggaagt
     3001 gacaggcgtc gtgttccatt atcgggcggc ccgggaccgc tatgcgctga ccttcgctga
     3061 ggcccaggag gcttgtcgcc taagctctgc taccatcgct gccccacggc acctgcaggc
     3121 tgcctttgaa gatggctttg acaactgcga cgcgggctgg ctctcagacc gcacggttcg
     3181 gtacccgatc actcagtcgc gtcctggttg ctatggtgac cgcagcagtc tcccgggtgt
     3241 tcggagctac gggagacgcg acccgcagga actctacgat gtctactgct ttgcccgcga
     3301 gctagggggt gaagtctttt acgtgggccc ggcccgccga ctgaccctgg cgggcgcgcg
     3361 ggcacaatgt cagcggcagg gtgcagcgct ggcctcggtg gggcagttgc acctggcctg
     3421 gcacgagggc ctggaccagt gcgacccggg ctggctggca gacggcagcg tgcgctaccc
     3481 catccagact ccgcgccggc gttgcggggg ccccgcccca ggtgtgcgca ctgtgtaccg
     3541 cttcgccaac cgcaccggct ttcctgcgcc aggagcacgc ttcgacgcct actgcttccg
     3601 agctcatcac catacagcac aacatggaga tctttaccct tacgatgtac cggattacgc
     3661 aggcggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     3721 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3781 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3841 gttcaggggg agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa
     3901 atcgataagg atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat
     3961 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4021 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4081 gagcgagcga gcgcgcagcc ttaattaacc taattcactg gccgtcgttt tacaacgtcg
     4141 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4201 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4261 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4321 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4381 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4441 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4501 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4561 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4621 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4681 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     4741 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4801 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4861 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4921 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4981 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5041 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5101 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5161 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5221 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5281 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5341 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5401 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5461 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5521 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5581 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5641 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5701 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5761 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5821 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5881 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5941 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6001 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6061 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6121 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6181 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6241 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6301 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6361 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6421 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6481 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6541 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     6601 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     6661 g
//
