LOCUS       sequence-439375-        6598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Secreted HA-tagged TurboID-NCAN-ELS domain for extracellular 
            proximity labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6598)
  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 6598)
  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 ELS-HA
FEATURES             Location/Qualifiers
     source          1..6598
                     /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             3572..3598
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3623..3744
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3634..3653)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3688..3707
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3906..4035
                     /label=AAV2 ITR
     primer_bind     complement(4054..4071)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4054..4070)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4063..4085)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4212..4667
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4299..4318)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4509..4530
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4693..4797
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4798..4866,4867..5658)
                     /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      5829..6417
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6318..6337
                     /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 catggaagag gtggccagcg ggcaggaaga
     2641 tcccacagat ccctgcgaga acaacccctg cctgcatggg ggcacctgcc acaccaacgg
     2701 caccgtgtat ggctgtagtt gtgaccaggg ctatgctggg gagaattgtg aaattgacat
     2761 tgatgactgc ttatgcagcc cttgcgagaa tgggggcacc tgcatcgacg aggtgaatgg
     2821 tttcatctgc ctctgcctcc ccagctatgg gggcagcctg tgtgagaagg acacagaagg
     2881 ctgcgaccgt ggctggcaca aattccaggg acactgctac cggtactttg cccatcgccg
     2941 ggcctgggag gatgcagaga gagattgcag gcgccgagct ggtcacctga caagcgtcca
     3001 ttcgccagaa gagcacaagt tcattaacag ttttggacat gagaattcgt ggatcggcct
     3061 gaatgaccgg acagtagaga gggacttcca gtggacagac aacacaggac tgcaatatga
     3121 gaactggagg gagaagcagc cggataattt cttcgcaggt ggggaggatt gtgtggtgat
     3181 ggtggcacat gagagtgggc gctggaatga tgtcccctgc aactacaacc taccctatgt
     3241 ctgcaagaag ggtacagtgc tgtgtggtcc ccctccagca gtggagaatg cctctcttgt
     3301 tggtgtgcgc aagatcaagt acaatgtcca tgccactgtg cgataccagt gtgatgaagg
     3361 attctcccag caccgagtgg ccactatccg ctgccgtaac aatgggaagt gggaccggcc
     3421 tcagatcatg tgcatcaaac ccaggcggtc acatcggatg cgtagacacc accaccatcc
     3481 acaccgacat cacaagcctc gcaaggagca cagaaaacac aagagacacc cagcggaaga
     3541 ctgggagaaa gacgaagggg atttctgcct ttacccttac gatgtaccgg attacgcagg
     3601 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     3661 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3721 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3781 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     3841 gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     3901 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3961 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4021 cgagcgagcg cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga
     4081 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4141 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4201 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4261 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4321 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4381 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4441 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4501 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4561 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4621 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     4681 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4741 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4801 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4861 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4921 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4981 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5041 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5101 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5161 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5221 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5281 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5341 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5401 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5461 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5521 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5581 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5641 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5701 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5761 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5821 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5881 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5941 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6001 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6061 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6121 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6181 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6241 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6301 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6361 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6421 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6481 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6541 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
