LOCUS       sequence-472208-        5775 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent ECS-facing destabilized SpyCatcher003 (facing the 
            extracellular space).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5775)
  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 5775)
  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-CAG-DIO-ECS-dSpyCatcher003
FEATURES             Location/Qualifiers
     source          1..5775
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(274..303)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(319..657)
                     /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"
     sig_peptide     complement(661..720)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      717..726
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(743..776)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(847..880)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    922..1510
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(975..995)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1393..1404)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    1553..1674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1564..1583)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1618..1637
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   1836..1965
                     /label=AAV2 ITR
     rep_origin      complement(2202..2790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2282..2301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2961..3752,3753..3821))
                     /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     3584..3603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3822..3926)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3952..4407)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4089..4110)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4301..4320
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   4479..4608
                     /label=AAV2 ITR
     promoter        4919..5196
                     /label=chicken beta-actin promoter
     primer_bind     5475..5497
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     5559..5578
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    5613..5646
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    5709..5742
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ctacacattg atcctagcag aagcacaggc tgcagggtga cggtccatcc cgctctcctg
       61 ggcacaagac atgggcagcg tgccatcatc ctgctcctcc acctccggcg ggaagccatg
      121 gctgcggggt ttcttttgcc acagcattat caaaataatc agtgatataa ttgtaagaac
      181 gacgagagcg agaattgcac ttataacgac gactttgaat ggcagtgaat gaggaacgac
      241 aataacttct tgagtatctt gtccgacggc gttgaggtct tcctcggaaa tcaacttctg
      301 ctcgctacca ctggaaccag tatgagcgtc accttcagtt gcttcaccat ctacagtaac
      361 ctgaccgtcc tcattaactg taaattcaat tggagttgct acctcataac cgtctggtgc
      421 tgcggtttcg acaaatgtat attttcctgg atacaggtag aaatccttca catgtccatc
      481 tgaaatccat gtactaatag ttttaccaga tgaatcacgc aactccatag ttgcaccagc
      541 taactcacgg ccgtcctcat cacgttttga gaatttaata tgggtagcac tatcttcttc
      601 agttgtcata tcaccggacg gaccttgctc acctgataaa cctgataagg tggttacgtc
      661 accagtggaa cctggaaccc agagcagcag tacccatagc aggagtgtgt ctgtctccat
      721 ggtggcggta ccgaattcta gtataacttc gtataggata ctttatacga agttatcatt
      781 gggattcttc ctattttgat ccaagcatca ccatcgaccc tctagtccag atctcaccat
      841 cgacccataa cttcgtatag catacattat acgaagttat gtccctcgaa gaggttcgaa
      901 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
      961 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     1021 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     1081 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     1141 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     1201 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     1261 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     1321 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     1381 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     1441 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     1501 gcctccccgc atcgataccg tcgacccggg cggccgcttc gagcagacat gataagatac
     1561 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     1621 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     1681 aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc
     1741 aagtaaaacc tctacaaatg tggtaaaatc gataaggatc ttcctagagc atggctacgt
     1801 agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc
     1861 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     1921 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc
     1981 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2041 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2101 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2161 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2221 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2281 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2341 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2401 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2461 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2521 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2581 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2641 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2701 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2761 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2821 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     2881 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2941 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3001 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3061 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3121 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3181 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3241 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3301 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3361 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3421 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3481 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3541 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3601 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3661 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3721 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3781 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     3841 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3901 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     3961 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4021 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4081 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4141 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4201 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4261 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4321 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4381 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     4441 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct gcgcgctcgc tcgctcactg
     4501 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     4561 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg tagttaatga
     4621 ttaacccgcc atgctactta tctacgtagc catgctctag gaagagtacc attgacgtca
     4681 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     4741 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     4801 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     4861 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
     4921 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
     4981 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
     5041 cgcgcgccag gggggggggg ggggggggag ggggggggsg gggsgaggsg gagaggtgcg
     5101 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
     5161 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
     5221 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
     5281 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
     5341 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
     5401 cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc ttcggggggg
     5461 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
     5521 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
     5581 gtctcatcat tttggcaaag aattggatcc ccataacttc gtataaagta tcctatacga
     5641 agttatatca aaataggaag accaatgctt caccatcgac ccgaattgcc aagcatcacc
     5701 atcgacccat aacttcgtat aatgtatgct atacgaagtt atactaggga tcccccctcg
     5761 agttttactt gtaca
//
