LOCUS       sequence-471546-        5968 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  to express SpyCatcher on the extracellular side of astrocytic 
            plasmamembrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5968)
  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 5968)
  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-SpyCatcher003
FEATURES             Location/Qualifiers
     source          1..5968
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      814..823
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     820..879
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             883..1221
                     /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             1237..1266
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    1434..2022
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1487..1507)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1905..1916)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2080..2099)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2134..2153
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   2249..2378
                     /label=AAV2 ITR
     rep_origin      complement(2868..3381)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3063..3084)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3275..3294
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3795..3814)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3914..3936
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3974..3992)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4060..4164
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4165..4233,4234..5025)
                     /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(4383..4402)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5196..5784
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5685..5704
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgcgcgc
       61 tcgctcgctc actgaggccg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg
      121 cgcagagagg gagtggccaa ctccatcact aggggttcct tgtagttaat gattaacccg
      181 ccatgctact tatctacgta gccatgctct agactcgaga cgcgtaacaa ataccatgtc
      241 gctggtatgg agtataggct gttgctatga caggaactca ggggtcttaa ctggcttgag
      301 cgctgggagg gggcaagcag ccaggccttg tctgtaagct gaagacctgg cagtgctgag
      361 ctggtcagcc cccaggacct ccttttgtgc ccacgagtga ctcaccttgg catagacata
      421 atggtcaggg gtgggcacgc agcctgcttc ccgctgtgct ccaggcctcc ttcgatgctt
      481 tccgagaagt ctattgagct gggagcttgt actgcacccg gggctgacat cctggcatcc
      541 tgggataaaa gcagcccacg gggctgccct tgccatatgc ctcactggcg gcagagaaca
      601 aggctctatt cagcgagtac cctggggagt tctcccccta gctgggctgg ggcccagctc
      661 caccccctca ggctattcaa tgggggtgct tccaggaagt caggggcaga tttagtccaa
      721 cccgttcctc cataaaggcc ctgacatccc aggagccagc agaggcaggg caggttggag
      781 cggagacgca tcacctctgc gcgccgcacc ggtgccacca tggagacaga cacactcctg
      841 ctatgggtac tgctgctctg ggttccaggt tccactggtg acgtaaccac cttatcaggt
      901 ttatcaggtg agcaaggtcc gtccggtgat atgacaactg aagaagatag tgctacccat
      961 attaaattct caaaacgtga tgaggacggc cgtgagttag ctggtgcaac tatggagttg
     1021 cgtgattcat ctggtaaaac tattagtaca tggatttcag atggacatgt gaaggatttc
     1081 tacctgtatc caggaaaata tacatttgtc gaaaccgcag caccagacgg ttatgaggta
     1141 gcaactccaa ttgaatttac agttaatgag gacggtcagg ttactgtaga tggtgaagca
     1201 actgaaggtg acgctcatac tggttccagt ggtagcgagc agaagttgat ttccgaggaa
     1261 gacctcaacg ccgtcggaca agatactcaa gaagttattg tcgttcctca ttcactgcca
     1321 ttcaaagtcg tcgttataag tgcaattctc gctctcgtcg ttcttacaat tatatcactg
     1381 attattttga taatgctgtg gcaaaagaaa ccccgctgag cggccgcatc gataatcaac
     1441 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     1501 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     1561 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     1621 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     1681 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     1741 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     1801 ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg
     1861 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     1921 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     1981 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatgc cgtatacggt
     2041 accacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     2101 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     2161 cttatcatgt ctgtcgaccc ccaacgcgtt ggagctctag agcatggcta cgtagataag
     2221 tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc
     2281 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     2341 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct ggcgtaatag cgaagagcca
     2401 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgattcca
     2461 acatccaata aatcatacag gcaaggcaaa gaattagcaa aattaagcaa taaagcctca
     2521 gagcataaag ctaaatcggt tgtaccaaaa acattatgac cctgtaatac ttttgcggga
     2581 gaagccttta tttcaacgca aggataaaaa tttttagaac cctcatatat tttaaatgca
     2641 atgcctgagt aatgtgtagg taaagattca aacgggtgag aaaggccgga gacagtcaaa
     2701 tcaccatcaa tatgatattc aaccgttcta gctgataaat tcatgccgga gagggtagct
     2761 atttttgaga ggtctctaca aaggctatca ggtcattgcc tgagagtctg gagcaaacaa
     2821 gagaatcgat gaacggtaat cgtaaaacta gcatgtcaat catatgtacc ccggttgata
     2881 atcagaaaag ccccaaaaac aggaagattg tataagcaaa tatttaaatt gtaagcgtta
     2941 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3001 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3061 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3121 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3181 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3241 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3301 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3361 atgcgccgct acagggcgcg tactatggtt gctttgacga gcacgtataa cgtgctttcc
     3421 tcgttagaat cagagcggga gctaaacagg aggccgatta aagggatttt agacaggaac
     3481 ggtacgccag aatcctgaga agtgttttta taatcagtga ggccaccgag taaaagagtc
     3541 tgtccatcac gcaaattaac cgttgtcgca atacttcttt gattagtaat aacatcactt
     3601 gcctgagtag aagaactcaa actatcggcc ttgctggtaa tatccagaac aatattaccg
     3661 ccagccattg caacggaatc gccattcgcc attcaggctg cgcaactgtt gawkggmggt
     3721 yggwgtcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3781 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     3841 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3901 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3961 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     4021 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4081 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4141 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4201 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4261 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4321 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4381 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4441 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4501 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4561 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4621 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4681 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4741 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4801 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4861 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4921 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4981 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5041 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5101 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5161 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5221 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5281 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5341 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5401 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5461 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5521 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5581 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5641 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5701 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5761 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5821 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5881 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     5941 gagtcagtga gcgaggaagc ggaagagc
//
