LOCUS       sequence-368405-        4941 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express the Clover4-LUMABS-DEN1 sensor for Dengue Virus antibody 
            dection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4941)
  AUTHORS   Deng M, Yuan J, Yang H, Wu X, Wei X, Du Y, Wong G, Tao Y, Liu G, Jin
            Z, Chu J
  TITLE     A Genetically Encoded Bioluminescent System for Fast and Highly 
            Sensitive Detection of Antibodies with a Bright Green Fluorescent 
            Protein.
  JOURNAL   ACS Nano. 2021 Nov 2. doi: 10.1021/acsnano.1c05164.
  PUBMED    34726889
REFERENCE   2  (bases 1 to 4941)
  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: "ACS Nano. 
            2021 Nov 2. doi: 10.1021/acsnano.1c05164."
COMMENT     Sequence Label: pNCS-Clover4-LUMABS-DEN1
FEATURES             Location/Qualifiers
     source          1..4941
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             122..144
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             164..181
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             185..217
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(221..229,230..244)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             263..280
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             290..307
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             428..439
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             join(563..565,566..568,569..1279)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFARYPDHMKLHDFFKAAMPEGYVQERTISFEDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKAKNGIK
                     ANFKIYHNVEDGSVQVADHYQQNTPIGDGPVLLPDNHYLSHQSTLSKDPNEKRDHMVLV
                     ERVTAAGITHGMDELYK"
     primer_bind     complement(608..629)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             1706..2218
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(2342..2360)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2356..2403
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2500..2955
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2587..2606)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2797..2818
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2981..3085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3086..3154,3155..3946)
                     /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(3304..3323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4117..4705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4606..4625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4859..4876
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaatcaccg agtttattct tgacacctga tgcgatgaat gatataatag gaaagtactg
      121 ttttgtttaa ctttaagaag gagatataca tatgcggggt tctcatcatc atcatcatca
      181 tggtatggct agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga
      241 taaggatccg atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg
      301 cggcagccat atggctagcg acgacaactt tatatacaag gcgaaggcgc tctatcccta
      361 tgatgcagat gatgatgacg catacgaaat ctcattcgaa caaaatgaaa tccttcaggt
      421 atcagacatt gaggggcggt ggtggaaagc ccgccgcgcc aatggagaga caggcatcat
      481 cccgtcgaat tatgttcagt tgattgacgg tcctgaggaa atgcatcggg gcggctcggg
      541 aggctcaggc agccatggat ccatggtgag caagggcgaa gagctgttca ccggggtggt
      601 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tccgcggcga
      661 gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa
      721 gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcgtgg cctgcttcgc
      781 caggtacccc gaccacatga agctgcacga cttcttcaag gccgccatgc ccgaaggcta
      841 cgtccaggag cgcaccatct ctttcgagga cgacggtacc tacaagaccc gcgccgaggt
      901 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
      961 ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca acgtctatat
     1021 catggccgac aaggccaaga acggcatcaa ggctaacttc aagatctacc acaacgttga
     1081 ggacggcagc gtgcaggtgg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1141 cgtgctgctg cccgacaacc actacctgag ccatcagtcc accctgagca aagaccccaa
     1201 cgagaagcgc gatcacatgg tcctggtgga gcgcgtgacc gccgccggga ttacacatgg
     1261 catggacgag ctgtacaagg aattctcggg cggacataaa tactcatgga agtcaggggg
     1321 ttcgggtggt tcaggcggct caggaggctc cgggggttcc ggagggagcg gtgctgaagc
     1381 cgcagccaag gaagcagcag ctaaagaggc cgctgcgaag gaagctgccg caaaggaggc
     1441 ggcggcgaaa gaggcggcag caaaagccgg atctggtggc agtggtggct ccggcgggtc
     1501 aggtggcagc gggggatcag gagctgaggc agccgccaaa gaggctgcgg ccaaggaggc
     1561 cgccgctaaa gaagccgcgg caaaagaggc agcggcaaag gaagcggctg cgaaagccgg
     1621 aagtggtggg tcgggcggct ccggtggctc tggcggcagt ggcggtagtg gcgggcacaa
     1681 gtatagctgg aaaagcggag gcagcatggt cttcacactc gaagatttcg ttggggactg
     1741 gcgacagaca gccggctaca acctggacca agtccttgaa cagggaggtg tgtccagttt
     1801 gtttcagaat ctcggggtgt ccgtaactcc gatccaaagg attgtcctga gcggtgaaaa
     1861 tgggctgaag atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg gcgaccaaat
     1921 gggccagatc gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc actttaaggt
     1981 gatcctgcac tatggcacac tggtaatcga cggggttacg ccgaacatga tcgactattt
     2041 cggacggccg tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg taacagggac
     2101 cctgtggaac ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg gctccctgct
     2161 gttccgagta accatcaacg gagtgaccgg ctggcggctg tgcgaacgca ttctggcgtc
     2221 tagcggtgga ggatctattc gttcaaagcc gctgccaccc ctacccgtga ccgggtaatg
     2281 aattcgaagc ttgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     2341 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     2401 ttgctgaaag gaggaactat atccggatct ggcgtaatag cgaagaggcc cgcaccgatc
     2461 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     2521 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2581 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2641 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2701 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2761 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2821 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2881 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     2941 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3001 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3061 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3121 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3181 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3241 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3301 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     3361 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3421 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3481 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3541 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3601 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     3661 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3721 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3781 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3841 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3901 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3961 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4021 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4081 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4141 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4201 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4261 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4321 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4381 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4441 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4501 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4561 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4621 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4681 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4741 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4801 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4861 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     4921 ttggccgatt cattaatgca g
//
