LOCUS       sequence-471025-        6488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  to express dSpyCatcher on the extracellular side of astrocytic 
            plasmamembrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6488)
  AUTHORS   Vittani M, Lee AB, Wang X, Hiraoka Y, Konno A, Knak PG, Kusk P, 
            Nagao M, Asiminas A, Courtin J, Putranto MF, Nasu Y, Tsuno S, Ueda 
            K, Osuga Y, Tsuboi T, Bienvenu T, Terunuma M, Hirai H, Nedergaard M,
            Tanaka K, Hirase H
  TITLE     Functional and structural profiling of circulation via genetically 
            encoded modular fluorescent probes
  JOURNAL   bioRxiv 2025.03.18.643859
REFERENCE   2  (bases 1 to 6488)
  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: "bioRxiv 
            2025.03.18.643859"
COMMENT     Sequence Label: pAAV-mGFAP(ABC1D)-ECS-dSpyCatcher003
FEATURES             Location/Qualifiers
     source          1..6488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(49..69)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(467..478)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    711..1105
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     repeat_region   1389..1518
                     /label=AAV2 ITR
     rep_origin      1889..2402
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1976..1995)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2186..2207
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2884..2903)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3003..3025
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3063..3081)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3149..3253
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3254..3322,3323..4114)
                     /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"
     primer_bind     complement(3472..3491)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4285..4873
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4774..4793
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     regulatory      5744..5753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     5750..5809
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             5813..6151
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     CDS             6167..6196
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    join(6484..6488,1..584)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
ORIGIN
        1 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
       61 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
      121 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
      181 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
      241 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
      301 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
      361 cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg
      421 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
      481 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
      541 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat gccgtatacg
      601 gtacccgggg atcctctagt agagtcgagg aattcacccc accagtgcag gctgcctatc
      661 agaaagtggt ggctggtgtg gctaatgccc tggcccacaa gtatcactaa gctcgctttc
      721 ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac taaactgggg
      781 gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt tattttcatt
      841 gcaatgatgt atttaaatta tttctgaata ttttactaaa aagggaatgt gggaggtcag
      901 tgcatttaaa acataaagaa atgaagagct agttcaaacc ttgggaaaat acactatatc
      961 ttaaactcca tgaaagaagg tgaggctgca aacagctaat gcacattggc aacagcccct
     1021 gatgcctatg ccttattcat ccctcagaaa aggattcaag tagaggcttg atttggaggt
     1081 taaagttttg ctatgctgta ttttacatta cttattgttt tagctgtcct catgaatgtc
     1141 ttttcactac ccatttgctt atcctgcatc tctcagcctt gactccactc agttctcttg
     1201 cttagagata ccacctttcc cctgaagtgt tccttccatg ttttacggcg agatggtttc
     1261 tcctcgcctg gccactcagc cttagttgtc tctgttgtct tatagaggtc tggtcgaccc
     1321 ccaacgcgtt ggagctctag agcatggcta cgtagataag tagcatggcg ggttaatcat
     1381 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     1441 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     1501 gagcgagcga gcgcgcagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     1561 acagttgcgc agcctgaatg gcgaatggcg attccgttgc aatggctggc ggtaatattg
     1621 ttctggatat taccagcaag gccgatagtt tgagttcttc tactcaggca agtgatgtta
     1681 ttactaatca aagaagtatt gcgacaacgg ttaatttgcg tgatggacag actcttttac
     1741 tcggtggcct cactgattat aaaaacactt ctcaggattc tggcgtaccg ttcctgtcta
     1801 aaatcccttt aatcggcctc ctgtttagct cccgctctga ttctaacgag gaaagcacgt
     1861 tatacgtgct cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     1921 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1981 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2041 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2101 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2161 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2221 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2281 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2341 atttaaatat ttgcttatac aatcttcctg tttttggggc ttttctgatt atcaaccggg
     2401 gtacatatga ttgacatgct agttttacga ttaccgttca tcgattctct tgtttgctcc
     2461 agactctcag gcaatgacct gatagccttt gtagagacct ctcaaaaata gctaccctct
     2521 ccggcatgaa tttatcagct agaacggttg aatatcatat tgatggtgat ttgactgtct
     2581 ccggcctttc tcacccgttt gaatctttac ctacacatta ctcaggcatt gcatttaaaa
     2641 tatatgaggg ttctaaaaat ttttatcctt gcgttgaaat aaaggcttct cccgcaaaag
     2701 tattacaggg tcataatgtt tttggtacaa ccgatttagc tttatgctct gaggctttat
     2761 tgcttaattt tgctaattct ttgccttgcc tgtatgattt attggatgtt ggaatcgcct
     2821 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     2881 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     2941 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3001 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     3061 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     3121 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3181 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3241 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3301 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3361 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3421 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3481 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3541 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3601 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3661 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3721 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3781 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3841 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3901 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3961 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4021 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     4081 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4141 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4201 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4261 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4321 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4381 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4441 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4501 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4561 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4621 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4681 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4741 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4801 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4861 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4921 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4981 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5041 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct tgtagttaat
     5101 gattaacccg ccatgctact tatctacgta gccatgctct agactcgaga cgcgtaacaa
     5161 ataccatgtc gctggtatgg agtataggct gttgctatga caggaactca ggggtcttaa
     5221 ctggcttgag cgctgggagg gggcaagcag ccaggccttg tctgtaagct gaagacctgg
     5281 cagtgctgag ctggtcagcc cccaggacct ccttttgtgc ccacgagtga ctcaccttgg
     5341 catagacata atggtcaggg gtgggcacgc agcctgcttc ccgctgtgct ccaggcctcc
     5401 ttcgatgctt tccgagaagt ctattgagct gggagcttgt actgcacccg gggctgacat
     5461 cctggcatcc tgggataaaa gcagcccacg gggctgccct tgccatatgc ctcactggcg
     5521 gcagagaaca aggctctatt cagcgagtac cctggggagt tctcccccta gctgggctgg
     5581 ggcccagctc caccccctca ggctattcaa tgggggtgct tccaggaagt caggggcaga
     5641 tttagtccaa cccgttcctc cataaaggcc ctgacatccc aggagccagc agaggcaggg
     5701 caggttggag cggagacgca tcacctctgc gcgccgcacc ggtgccacca tggagacaga
     5761 cacactcctg ctatgggtac tgctgctctg ggttccaggt tccactggtg acgtaaccac
     5821 cttatcaggt ttatcaggtg agcaaggtcc gtccggtgat atgacaactg aagaagatag
     5881 tgctacccat attaaattct caaaacgtga tgaggacggc cgtgagttag ctggtgcaac
     5941 tatggagttg cgtgattcat ctggtaaaac tattagtaca tggatttcag atggacatgt
     6001 gaaggatttc tacctgtatc caggaaaata tacatttgtc gaaaccgcag caccagacgg
     6061 ttatgaggta gcaactccaa ttgaatttac agttaatgag gacggtcagg ttactgtaga
     6121 tggtgaagca actgaaggtg acgctcatac tggttccagt ggtagcgagc agaagttgat
     6181 ttccgaggaa gacctcaacg ccgtcggaca agatactcaa gaagttattg tcgttcctca
     6241 ttcactgcca ttcaaagtcg tcgttataag tgcaattctc gctctcgtcg ttcttacaat
     6301 tatatcactg attattttga taatgctgtg gcaaaagaaa ccccgcagcc atggcttccc
     6361 gccggaggtg gaggagcagg atgatggcac gctgcccatg tcttgtgccc aggagagcgg
     6421 gatggaccgt caccctgcag cctgtgcttc tgctaggatc aatgtgtagg cggccgcatc
     6481 gataatca
//
