LOCUS       sequence-437697-        6255 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses the 18-kDa Large BiT (LB) fragment of split NanoLuc, 
            genetically fused via a semi-rigid peptide linker to a protein G 
            adapter domain (Gx)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6255)
  AUTHORS   Ni Y, Rosier BJHM, van Aalen EA, Hanckmann ETL, Biewenga L, Pistikou
            AM, Timmermans B, Vu C, Roos S, Arts R, Li W, de Greef TFA, van 
            Borren MMGJ, van Kuppeveld FJM, Bosch BJ, Merkx M
  TITLE     A plug-and-play platform of ratiometric bioluminescent sensors for 
            homogeneous immunoassays.
  JOURNAL   Nat Commun. 2021 Jul 28;12(1):4586. doi: 10.1038/s41467-021-24874-3.
  PUBMED    34321486
REFERENCE   2  (bases 1 to 6255)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-07-28"; volume: "12(1):4586. doi"; pages: " 
            10.1038/s41467-021-24874-3"
COMMENT     Sequence Label: pET28a(+)-Gx-LB
FEATURES             Location/Qualifiers
     source          1..6255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             350..541
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(535..557)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    643..782
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     698..717
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(797..814)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(968..1556)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1048..1067)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1667..1686)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1678..2493
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1678..2493
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1751..1770)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2585..3040)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2722..2743)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2934..2953
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3077..3124)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3120..3138
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3191..3208)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3227..3244)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3296..3313)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4211..4234)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     RBS             4247..4269
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4284..4308
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(4308..4327)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4309..4327)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4411..4430)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4636..4713
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4714..5796
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4733..4752)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
       61 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
      121 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
      181 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
      241 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
      301 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
      361 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
      421 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
      481 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
      541 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
      601 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
      661 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
      721 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
      781 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
      841 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
      901 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
      961 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1021 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1081 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1141 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1201 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1261 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     1321 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     1381 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     1441 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     1501 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     1561 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     1621 ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     1681 agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct
     1741 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     1801 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     1861 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     1921 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     1981 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     2041 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     2101 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     2161 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     2221 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     2281 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     2341 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     2401 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     2461 cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt
     2521 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     2581 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2641 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     2701 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     2761 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     2821 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     2881 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     2941 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3001 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc aatccggata
     3061 tagttcctcc tttcagcaaa aaacccctca agacccgttt agaggcccca aggggttatg
     3121 ctagttattg ctcagcggtg gcagcagcca actcagcttc ctttcgggct ttgttagcag
     3181 ccggatctca gtggtggtgg tggtggtgct cgagtgcggc cgcttaatgg tggtgatgat
     3241 gatgtgaacc gccgctctct ttttcaaaca gacgatagcc ggtaacggta ccttagtggt
     3301 gatggtggtg gtgggaacct ccgctgttga tggttactcg gaacagcatg gagccgtcgg
     3361 gggtgatcag gcgctcgtcg ataattttgt tgccgttcca cagggtccct gttacagtga
     3421 tctttttgcc gtcgaacacg gcgatgcctt catacggccg tccgaaatag ttcagcatgt
     3481 tcggcgtaac cccgtcgatt accagtgtgc catagggcag gatcacctta aagtgatgat
     3541 catccacagg gtacaccacc ttaaacacct cttcgatctg ggccatttgg tcggcgctca
     3601 gaccttcata cgggatgatg acatggatgt cgatcttcag ggcattttca ccgctccgga
     3661 caatcctttg gatcggagtt acggacacgg cgagattctg cagcaaactg gacacacctc
     3721 cctgttcaag gacttggtcc aggttgtagg cggctgtctg ttcccagtcc ccaacgaaat
     3781 cttcgagtgt gaagacggta cccccagaac cgcctgaacc cccgcttcca ccactacccc
     3841 ccgagccgcc gctacccccg aattctgcct tggctgctgc ttccttcgcc gcggcctctt
     3901 ttgcggctgc ctccttggcc gcggcttcct ttgctgcggc ttctttagcg gctgcttcgg
     3961 cgaattcacc gctaccacca cttcctccag agccgccaga acctcccgac ccacccgaac
     4021 ctcctgtaaa ttcttctgtt accgtgaaag ttttagttgc gtcgtcataa gtccattcac
     4081 catcaattcc ataatcattt gcatactgct taaaaatctt ctccgcctcc taagcatcca
     4141 ccgcttctat tgtgatctct ccctttaaag ttttgccgtt gataatcagt ttaaatgtca
     4201 tgctaccacc tttttcaaac tgcggatggc tccagcccat ggtatatctc cttcttaaag
     4261 ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt
     4321 cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca
     4381 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     4441 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
     4501 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
     4561 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
     4621 agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
     4681 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     4741 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     4801 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     4861 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     4921 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     4981 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     5041 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     5101 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     5161 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     5221 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     5281 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     5341 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     5401 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     5461 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     5521 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     5581 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     5641 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     5701 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5761 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     5821 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag
     5881 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     5941 catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt
     6001 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct
     6061 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat
     6121 cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     6181 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     6241 aagcgactgc tgctg
//
