LOCUS       sequence-471023-        5469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  to express a green fluorescent protein tagged with SpyCatcher in the
            liver.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5469)
  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 5469)
  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-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..5469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             61..399
                     /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             415..1122
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1167..1755
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1220..1240)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1638..1649)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    1773..1997
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2042..2182
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2042..2171
                     /label=AAV2 ITR
     rep_origin      2257..2712
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2344..2363)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2554..2575
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2729..2748)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2848..2870
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2908..2926)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2994..3098
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3099..3167,3168..3959)
                     /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(3317..3336)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4130..4718
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4619..4638
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   4955..5084
                     /label=AAV2 ITR
ORIGIN
        1 atgacagaca cactcctgct atgggtactg ctgctctggg ttccaggttc cactggtgac
       61 gttactacac tcagtggact ttccggagaa cagggcccaa gtggggacat gaccacagaa
      121 gaggactccg ccacacacat caagttttcc aagagagacg aagatggaag ggaattggcc
      181 ggcgctacca tggaactcag ggacagctcc gggaagacaa tctccacttg gatcagtgac
      241 ggccacgtaa aagactttta tctttaccct gggaagtaca ctttcgtgga gacagccgcc
      301 cccgatggct acgaagtggc cacaccgatc gagttcacgg tcaacgaaga tggccaagtg
      361 acagtggacg gcgaggccac cgagggcgat gcccacacag ggagctctgg atcagtgagc
      421 aagggcgagg aggataacat ggcctctctc ccagcgacac atgagttaca catctttggc
      481 tccatcaacg gtgtggactt tgacatggtg ggtcagggca ccggcaatcc aaatgatggt
      541 tatgaggagt taaacctgaa gtccaccaag ggtgacctcc agttctcccc ctggattctg
      601 gtccctcata tcgggtatgg cttccatcag tacctgccct accctgacgg gatgtcgcct
      661 ttccaggccg ccatggtaga tggctccggc taccaagtcc atcgcacaat gcagtttgaa
      721 gatggtgcct cccttactgt taactaccgc tacacctacg agggaagcca catcaaagga
      781 gaggcccagg tgaaggggac tggtttccct gctgacggtc ctgtgatgac caactcgctg
      841 accgctgcgg actggtgcag gtcgaagaag acttacccca acgacaaaac catcatcagt
      901 acctttaagt ggagttacac cactggaaat ggcaagcgct accggagcac tgcgcggacc
      961 acctacacct ttgccaagcc aatggcggct aactatctga agaaccagcc gatgtacgtg
     1021 ttccgtaaga cggagctcaa gcactccaag accgagctca acttcaagga gtggcaaaag
     1081 gcctttaccg atgtgatggg catggacgag ctgtacaagt aagcggccgc aagctttcta
     1141 gagaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     1201 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     1261 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     1321 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     1381 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     1441 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     1501 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     1561 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     1621 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     1681 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     1741 gggccgcctc cccgctctcg agatacgtac gactgtgcct tctagttgcc agccatctgt
     1801 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1861 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     1921 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     1981 tgcggtgggc tctatggctt ctgaggcgga aagatctacc acgtgcggac cgagcggccg
     2041 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     2101 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     2161 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     2221 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     2281 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     2341 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2401 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2461 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2521 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2581 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     2641 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2701 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     2761 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     2821 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     2881 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     2941 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3001 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3061 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3121 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3181 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3241 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3301 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     3361 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3421 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3481 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3541 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3601 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     3661 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     3721 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     3781 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     3841 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     3901 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     3961 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4021 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4081 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4141 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4201 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4261 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4321 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4381 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4441 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4501 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4561 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4621 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4681 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4741 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4801 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4861 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     4921 ctctccccgc gcgttggccg attcattaat gcagctgcgc gctcgctcgc tcactgaggc
     4981 cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
     5041 agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt taatgattaa
     5101 cccgccatgc tacttatcta ctcgagaccg ggcgcgccgg gggaggctgc tggtgaatat
     5161 taaccaaggt caccccagtt atcggaggag caaacagggg ctaagtccac acgcgtggta
     5221 ccgtctgtct gcacatttcg tagagcgagt gttccgatac tctaatctcc ctaggcaagg
     5281 ttcatatttg tgtaggttac ttattctcct tttgttgact aagtcaataa tcagaatcag
     5341 caggtttgga gtcagcttgg cagggatcag cagcctgggt tggaaggagg gggtataaaa
     5401 gccccttcac caggagaagc cgtcacacag atccacaagc tcctggtcga cactagtacc
     5461 ggtgccacc
//
