LOCUS       sequence-368398-        5247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express the Clover4-LUMABS-VCA sensor for Epstein-barr Virus 
            antibody dection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5247)
  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 5247)
  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-VCA
FEATURES             Location/Qualifiers
     source          1..5247
                     /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             2012..2524
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(2648..2666)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2662..2709
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2806..3261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2893..2912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3103..3124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3287..3391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3392..3460,3461..4252)
                     /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(3610..3629)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4423..5011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4912..4931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5165..5182
                     /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 cggaagcacc gcggttgcgc aaagcgcgac
     1321 cccgagcgtt agcagcagca ttagcagcct gcgtgcggcg accagcggtg cgaccgcggc
     1381 ggcgagcgcg gcggcggcgg tggataccgg tagcggtggt ggcggtcaac cgcacgacac
     1441 cgcgccgcgt ggtgcgcgta aaaagcaggg gggttcgggt ggttcaggcg gctcaggagg
     1501 ctccgggggt tccggaggga gcggtgctga agccgcagcc aaggaagcag cagctaaaga
     1561 ggccgctgcg aaggaagctg ccgcaaagga ggcggcggcg aaagaggcgg cagcaaaagc
     1621 cggatctggt ggcagtggtg gctccggcgg gtcaggtggc agcgggggat caggagctga
     1681 ggcagccgcc aaagaggctg cggccaagga ggccgccgct aaagaagccg cggcaaaaga
     1741 ggcagcggca aaggaagcgg ctgcgaaagc cggaagtggt gggtcgggcg gctccggtgg
     1801 ctctggcggc agtggcggta gtggcgggag caccgcggtg gcgcagagcg cgaccccgag
     1861 cgtgagcagc agcatcagca gcctgcgtgc ggcgaccagc ggcgcgaccg cggcggcgag
     1921 cgcggcggcg gcggttgaca ccggtagcgg cggtggcggt cagccgcacg ataccgcgcc
     1981 gcgtggcgcg cgtaagaaac aaggaggcag catggtcttc acactcgaag atttcgttgg
     2041 ggactggcga cagacagccg gctacaacct ggaccaagtc cttgaacagg gaggtgtgtc
     2101 cagtttgttt cagaatctcg gggtgtccgt aactccgatc caaaggattg tcctgagcgg
     2161 tgaaaatggg ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcggcga
     2221 ccaaatgggc cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg atcatcactt
     2281 taaggtgatc ctgcactatg gcacactggt aatcgacggg gttacgccga acatgatcga
     2341 ctatttcgga cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac
     2401 agggaccctg tggaacggca acaaaattat cgacgagcgc ctgatcaacc ccgacggctc
     2461 cctgctgttc cgagtaacca tcaacggagt gaccggctgg cggctgtgcg aacgcattct
     2521 ggcgtctagc ggtggaggat ctattcgttc aaagccgctg ccacccctac ccgtgaccgg
     2581 gtaatgaatt cgaagcttga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     2641 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     2701 ggttttttgc tgaaaggagg aactatatcc ggatctggcg taatagcgaa gaggcccgca
     2761 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg
     2821 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2881 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2941 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3001 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3061 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3121 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3181 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3241 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3301 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3361 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3421 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3481 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3541 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3601 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3661 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3721 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3781 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3841 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3901 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3961 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4021 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4081 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4141 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4201 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4261 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4321 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4381 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4441 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4501 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4561 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4621 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4681 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4741 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4801 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4861 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4921 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4981 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5041 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5101 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5161 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     5221 cgcgcgttgg ccgattcatt aatgcag
//
