LOCUS       sequence-437695-        5745 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses the 1.3-kDa Small BiT (SB) 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 5745)
  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 5745)
  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-SB
FEATURES             Location/Qualifiers
     source          1..5745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             90..281
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(275..297)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    383..522
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     438..457
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(537..554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(708..1296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(788..807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1407..1426)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1418..2233
                     /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             1418..2233
                     /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(1491..1510)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2325..2780)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2462..2483)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2674..2693
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2817..2864)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2860..2878
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2931..2948)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2967..2984)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3441..3464)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     RBS             3477..3499
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3514..3538
                     /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(3538..3557)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3539..3557)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3641..3660)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3866..3943
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3944..5026
                     /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(3963..3982)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga
       61 acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca
      121 tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg
      181 cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca
      241 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
      301 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
      361 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
      421 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt
      481 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
      541 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
      601 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      661 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      721 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      781 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      841 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      901 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      961 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1021 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1081 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1141 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1201 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1261 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1321 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa
     1381 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     1441 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     1501 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     1561 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     1621 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     1681 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca
     1741 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     1801 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     1861 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     1921 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     1981 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     2041 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     2101 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     2161 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     2221 tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata
     2281 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     2341 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     2401 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     2461 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     2521 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     2581 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     2641 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     2701 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     2761 tgcgccgcta cagggcgcgt cccattcgcc aatccggata tagttcctcc tttcagcaaa
     2821 aaacccctca agacccgttt agaggcccca aggggttatg ctagttattg ctcagcggtg
     2881 gcagcagcca actcagcttc ctttcgggct ttgttagcag ccggatctca gtggtggtgg
     2941 tggtggtgct cgagtgcggc cgcttaatgg tggtgatgat gatgtgaacc gccgctctct
     3001 ttttcaaaca gacgatagcc ggtaacggta cccccagaac cgcctgaacc cccgcttcca
     3061 ccactacccc ccgagccgcc gctacccccg aattctgcct tggctgctgc ttccttcgcc
     3121 gcggcctctt ttgcggctgc ctccttggcc gcggcttcct ttgctgcggc ttctttagcg
     3181 gctgcttcgg cgaattcacc gctaccacca cttcctccag agccgccaga acctcccgac
     3241 ccacccgaac ctcctgtaaa ttcttctgtt accgtgaaag ttttagttgc gtcgtcataa
     3301 gtccattcac catcaattcc ataatcattt gcatactgct taaaaatctt ctccgcctcc
     3361 taagcatcca ccgcttctat tgtgatctct ccctttaaag ttttgccgtt gataatcagt
     3421 ttaaatgtca tgctaccacc tttttcaaac tgcggatggc tccagcccat ggtatatctc
     3481 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
     3541 tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc
     3601 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     3661 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     3721 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     3781 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     3841 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
     3901 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     3961 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     4021 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     4081 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     4141 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     4201 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     4261 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     4321 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     4381 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     4441 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     4501 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     4561 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     4621 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     4681 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     4741 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     4801 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     4861 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     4921 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     4981 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     5041 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca
     5101 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     5161 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg
     5221 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct
     5281 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc
     5341 aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga
     5401 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg
     5461 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg
     5521 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta
     5581 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     5641 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg
     5701 ccagttgttt accctcacaa cgttccagta accgggcatg ttcat
//
