LOCUS       sequence-368401-        5253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express the Clover4-LUMABS-EBNA1 sensor for Epstein-barr Virus 
            antibody dection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5253)
  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 5253)
  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-EBNA1
FEATURES             Location/Qualifiers
     source          1..5253
                     /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             2018..2530
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(2654..2672)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2668..2715
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2812..3267
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2899..2918)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3109..3130
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3293..3397
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3398..3466,3467..4258)
                     /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(3616..3635)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4429..5017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4918..4937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5171..5188
                     /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 cggaccgccg cgtcgtccgc cgccgggtcg
     1321 tcgtccgttc ttccacccgg ttggcgaagc ggactatttc gagtaccacc aagaaggtgg
     1381 cccggatggc gaaccggatg ttccgccggg cgcgatcgaa cagggtccgg cggatgaccc
     1441 gggtgaaggt ccgagcaccg gcccgcgtgg tgggggttcg ggtggttcag gcggctcagg
     1501 aggctccggg ggttccggag ggagcggtgc tgaagccgca gccaaggaag cagcagctaa
     1561 agaggccgct gcgaaggaag ctgccgcaaa ggaggcggcg gcgaaagagg cggcagcaaa
     1621 agccggatct ggtggcagtg gtggctccgg cgggtcaggt ggcagcgggg gatcaggagc
     1681 tgaggcagcc gccaaagagg ctgcggccaa ggaggccgcc gctaaagaag ccgcggcaaa
     1741 agaggcagcg gcaaaggaag cggctgcgaa agccggaagt ggtgggtcgg gcggctccgg
     1801 tggctctggc ggcagtggcg gtagtggcgg gccgccgcgt cgtccgccgc cgggccgtcg
     1861 tccgtttttc cacccggtgg gtgaagcgga ttacttcgaa taccaccagg aaggcggtcc
     1921 ggatggcgag ccggacgttc cgccgggtgc gattgagcaa ggcccggcgg acgatccggg
     1981 tgaaggcccg agcaccggtc cgcgtggtgg aggcagcatg gtcttcacac tcgaagattt
     2041 cgttggggac tggcgacaga cagccggcta caacctggac caagtccttg aacagggagg
     2101 tgtgtccagt ttgtttcaga atctcggggt gtccgtaact ccgatccaaa ggattgtcct
     2161 gagcggtgaa aatgggctga agatcgacat ccatgtcatc atcccgtatg aaggtctgag
     2221 cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca
     2281 tcactttaag gtgatcctgc actatggcac actggtaatc gacggggtta cgccgaacat
     2341 gatcgactat ttcggacggc cgtatgaagg catcgccgtg ttcgacggca aaaagatcac
     2401 tgtaacaggg accctgtgga acggcaacaa aattatcgac gagcgcctga tcaaccccga
     2461 cggctccctg ctgttccgag taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg
     2521 cattctggcg tctagcggtg gaggatctat tcgttcaaag ccgctgccac ccctacccgt
     2581 gaccgggtaa tgaattcgaa gcttgatccg gctgctaaca aagcccgaaa ggaagctgag
     2641 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     2701 ttgaggggtt ttttgctgaa aggaggaact atatccggat ctggcgtaat agcgaagagg
     2761 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
     2821 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2881 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2941 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3001 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3061 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3121 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3181 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3241 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     3301 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3361 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3421 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3481 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3541 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3601 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3661 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3721 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3781 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3841 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3901 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3961 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4021 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4081 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     4141 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4201 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     4261 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4321 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4381 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4441 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4501 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4561 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4621 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4681 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4741 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4801 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4861 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4921 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4981 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5041 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5101 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     5161 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     5221 tctccccgcg cgttggccga ttcattaatg cag
//
