LOCUS       sequence-471548-        5928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  to express a potassium sensor tagged with SpyCatcher in the liver.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5928)
  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 5928)
  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-GINKO2
FEATURES             Location/Qualifiers
     source          1..5928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     sig_peptide     1..60
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             64..402
                     /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    1626..2214
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1679..1699)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2097..2108)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2232..2456
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2501..2641
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2501..2630
                     /label=AAV2 ITR
     rep_origin      2716..3171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2803..2822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3013..3034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3188..3207)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3307..3329
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3367..3385)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3453..3557
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3558..3626,3627..4418)
                     /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(3776..3795)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4589..5177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5078..5097
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5414..5543
                     /label=AAV2 ITR
     regulatory      join(5923..5928,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt
       61 gacgttacaa ctctgtctgg cttgtctggc gaacaaggtc ctagtggcga catgaccacc
      121 gaggaagact ctgcgacaca catcaagttt agcaagcgcg acgaagatgg gagagaactg
      181 gcgggagcca caatggaact gcgggacagc tccggcaaga ccatcagcac ctggatcagc
      241 gacggccacg taaaagactt ttatctttac cctgggaagt acaccttcgt ggagacagcg
      301 gcccctgatg gatacgaagt tgctacgcct atcgagttca ccgtgaacga agatggacaa
      361 gtcaccgtgg acggagaggc caccgaaggg gatgcacaca ccggcagtag cggatcagtc
      421 tcaaaagggg aagaactctt tactggcgtc gttccaatac tagtggaatt ggatggtgat
      481 gtgaatgggc ataaattttc agtctatgga gaaggagaag gtgacgcgac atatggtaaa
      541 ctcactctca aatttatttg tacaactggt aaattgccag tcccttggcc taccttggta
      601 acgacactta catatggagt acaatgcttt tctcggtatc ctgatcatat gaaacaacat
      661 gatttcttta aaagtgcgat gcctgagggt tatacgcagg aaagaacaat cttcttcgaa
      721 gatgatggga attataaaac aagggcagaa gttaaatttg aaggagatac actcgttaat
      781 cgcattgaac tggaagggat tgattttaaa gaagacggga atatactcgg ccataaattg
      841 gaatacaatt tgccagatgg tttgtttaat tttgttaaag atgctgggga gaaactctgg
      901 gacgcagtca cgggacaaca tgataaagat gatcaagcta agaaagtctg ggagcatctc
      961 cataaaacag gaataccaga tgcagataag gttaatatac aagtggcgga tgggaaagca
     1021 actgttgcgg gcgatggtct ctctcaagag gcaaaggaga aaattctcgt tgcagtcgga
     1081 aatattagtg gcattgcatc tgttgatgat caggtaaaga cagcaacgcc cgctactgct
     1141 tctcaatttt atactgtcaa atctggagat acactatccg caatcagcaa ccaagtctat
     1201 ggaaatgctg atctctataa taaaatcttt gaagccaata aaccaatgtt gaagtctcct
     1261 gataagattt atcctgggca agttctccgg attccttacg cagcaaacaa cgtgtacatt
     1321 aaagcggata aagaaaagaa tggtattaaa gccaatttta agataaggca taatatagaa
     1381 gatggcggag tccagctagc gtatcattat caacaaaata caccaattgg agatggacca
     1441 gtcttgctcc cggataatca ttatctttca gtccaaagta ggctctcaaa agatccaaat
     1501 gaaaagcggg accatatggt tctactcgaa tttgtcacgg ctgctggaat tacactggga
     1561 atggatgaac tctatatgta ggcggccgca agctttctag agaattcgat atcaagctta
     1621 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     1681 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     1741 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     1801 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     1861 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     1921 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     1981 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     2041 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     2101 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     2161 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgctctcga
     2221 gatacgtacg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2281 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2341 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2401 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2461 tgaggcggaa agatctacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     2521 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     2581 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     2641 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     2701 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2761 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     2821 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2881 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     2941 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3001 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     3061 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     3121 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     3181 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     3241 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3301 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     3361 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3421 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3481 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3541 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3601 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3661 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3721 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3781 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     3841 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3901 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3961 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4021 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4081 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4141 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4201 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4261 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4321 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4381 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4441 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4501 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4561 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4621 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4681 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4741 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4801 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4861 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4921 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4981 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5041 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5101 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5161 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5221 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5281 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5341 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     5401 ttcattaatg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg
     5461 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc
     5521 caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac
     5581 tcgagaccgg gcgcgccggg ggaggctgct ggtgaatatt aaccaaggtc accccagtta
     5641 tcggaggagc aaacaggggc taagtccaca cgcgtggtac cgtctgtctg cacatttcgt
     5701 agagcgagtg ttccgatact ctaatctccc taggcaaggt tcatatttgt gtaggttact
     5761 tattctcctt ttgttgacta agtcaataat cagaatcagc aggtttggag tcagcttggc
     5821 agggatcagc agcctgggtt ggaaggaggg ggtataaaag ccccttcacc aggagaagcc
     5881 gtcacacaga tccacaagct cctggtcgac actagtaccg gtgccacc
//
