LOCUS       sequence-471017-        5478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  to express a pH sensor tagged with SpyCatcher in the liver.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5478)
  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 5478)
  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-P3-IgKL-SpyCatcher003-SEP
FEATURES             Location/Qualifiers
     source          1..5478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(12..23)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    147..371
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   416..556
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   416..545
                     /label=AAV2 ITR
     rep_origin      631..1086
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(718..737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     928..949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1103..1122)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1222..1244
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1282..1300)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1368..1472
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1473..1541,1542..2333)
                     /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(1691..1710)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2504..3092
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2993..3012
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3329..3458
                     /label=AAV2 ITR
     regulatory      3838..3847
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     3844..3903
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             3907..4245
                     /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"
     misc_feature    join(5019..5478,1..129)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5072..5092)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
       61 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
      121 cctccccgct ctcgagatac gtacgactgt gccttctagt tgccagccat ctgttgtttg
      181 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
      241 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
      301 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
      361 gggctctatg gcttctgagg cggaaagatc taccacgtgc ggaccgagcg gccgcaggaa
      421 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
      481 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
      541 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
      601 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
      661 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
      721 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      781 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      841 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      901 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      961 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     1021 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     1081 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     1141 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     1201 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     1261 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1321 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1381 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1441 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1501 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1561 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1621 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1681 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1741 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1801 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1861 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1921 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1981 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     2041 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2101 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2161 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     2221 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2281 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2341 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2401 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2461 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2521 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2581 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2641 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2701 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2761 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2821 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2881 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2941 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3001 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3061 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3121 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3181 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3241 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     3301 ccgcgcgttg gccgattcat taatgcagct gcgcgctcgc tcgctcactg aggccgcccg
     3361 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     3421 cagagaggga gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc
     3481 atgctactta tctactcgag accgggcgcg ccgggggagg ctgctggtga atattaacca
     3541 aggtcacccc agttatcgga ggagcaaaca ggggctaagt ccacacgcgt ggtaccgtct
     3601 gtctgcacat ttcgtagagc gagtgttccg atactctaat ctccctaggc aaggttcata
     3661 tttgtgtagg ttacttattc tccttttgtt gactaagtca ataatcagaa tcagcaggtt
     3721 tggagtcagc ttggcaggga tcagcagcct gggttggaag gagggggtat aaaagcccct
     3781 tcaccaggag aagccggcac acagatccac aagctcctgg tcgacactag taccggtgcc
     3841 accatggaga cagacacact cctgctatgg gtactgctgc tctgggttcc aggttccact
     3901 ggtgacgtca ctacactaag cggcctgtca ggcgaacaag gacccagcgg agacatgact
     3961 accgaagagg acagcgcgac acacatcaag ttttccaagc gggacgaaga tggaagagaa
     4021 cttgcgggag ctaccatgga actacgagac agttcaggga agacaatctc aacctggatc
     4081 tccgacggcc acgttaaaga cttttatctc taccctggta agtacacgtt cgtggagact
     4141 gccgcccccg atggatacga agtggcgacc cccatcgagt tcaccgtaaa cgaagatgga
     4201 caagtgacag tggacggcga ggccacagag ggcgatgcac acacaggcag ctcaggcagt
     4261 agcaagggtg aggagctgtt tacaggcgtg gtacctatct tggtagagct tgacggagac
     4321 gtcaacggcc ataagttctc cgtgagcggc gaaggggaag gggacgctac ctatgggaag
     4381 ctcactctaa agttcatatg taccaccggg aagctgcccg tgccgtggcc tactttggtt
     4441 accaccctga cctacggagt ccagtgtttc agtcgctatc ccgaccacat gaagagacac
     4501 gatttcttta aaagcgctat gcctgaggga tacgtgcaag agcgcacaat cttctttaag
     4561 gacgatggca attataaaac ccgagcagag gtgaaattcg aaggggacac tttggtgaac
     4621 aggattgaac tgaaggggat cgacttcaag gaagacggca atatcctcgg tcataagctg
     4681 gagtataact acaatgacca tcaagtctat attatggctg ataagcagaa gaacggtatt
     4741 aaggccaatt ttaagatccg acataatatc gaggacggcg gtgtgcagct ggctgatcac
     4801 taccagcaga acacgcccat cggagacgga ccagtgctct tgcccgataa tcactatctc
     4861 ttcaccacca gcaccctgag caaggaccct aatgagaaaa gggatcatat ggtgctcctg
     4921 gaatttgtca ccgccgccgg tatcacccac ggaatggacg agctgtataa gtgagcggcc
     4981 gcaagctttc tagagaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     5041 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     5101 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     5161 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     5221 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     5281 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     5341 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     5401 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     5461 tgcgcgggac gtccttct
//
