LOCUS       sequence-335658-        6039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses protein G fused to NanoLuc. The protein G domain contains 
            an amber stop-codon at position 24 for incorporation of the 
            unnatural amino acid para-benzoylphenylalanine..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6039)
  AUTHORS   Wouters SFA, Vugs WJP, Arts R, de Leeuw NM, Teeuwen RWH, Merkx M
  TITLE     Bioluminescent Antibodies through Photoconjugation of Protein 
            G-Luciferase Fusion Proteins.
  JOURNAL   Bioconjug Chem. 2020 Mar 18;31(3):656-662. doi: 
            10.1021/acs.bioconjchem.9b00804. Epub 2020 Jan 22.
  PUBMED    31909607
REFERENCE   2  (bases 1 to 6039)
  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"; doi: 
            "10.1021/acs.bioconjchem.9b00804"; journalName: "Bioconjug Chem"; 
            date: "2020-03-18- 18"; volume: "31"; issue: "3"; pages: "656-662"
COMMENT     Sequence Label: pET28a Gx-NL
FEATURES             Location/Qualifiers
     source          1..6039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     549..568
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(588..665)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     871..890
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     974..993
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        974..992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    993..1017
                     /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)."
     RBS             1032..1054
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1067..1090
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1850..1867
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1877..1894
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1947..1965)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1961..2008
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2045..2500
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2132..2151)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2342..2363
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2592..3407)
                     /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             complement(2592..3407)
                     /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     3315..3334
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3399..3418
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3529..4117
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4018..4037
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4271..4288
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4303..4442
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4368..4387)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4528..4550
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4544..4735)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5544..6039,1..587))
                     /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"
ORIGIN
        1 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
       61 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      121 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      181 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      241 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      301 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      361 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      421 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      481 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      541 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
      601 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
      661 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
      721 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
      781 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
      841 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
      901 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
      961 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1021 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggctggt cccatccgca
     1081 gttcgagaaa ggtaccatga catttaaact gataatcaac ggcaaaacct taaaagggga
     1141 gatcacaatt gaggcagtcg atgcctagga agccgagaaa atctttaaac aatatgctaa
     1201 tgattatggt attgacggag aatggacgta tgacgatgcg acaaaaactt tcaccgtaac
     1261 tgaggaattc actagtggtg gaagtggggg cagcatggta ttcacattgg aagatttcgt
     1321 gggtgattgg cgacaaacgg ccggttacaa tttggatcag gtgttagaac aagggggcgt
     1381 aagctccctg ttccagaatt taggagtgag cgtgacacct attcagcgca ttgtgctgag
     1441 cggcgaaaat ggtctgaaaa ttgatattca tgtgatcatc ccttacgaag gcctgtctgg
     1501 ggatcaaatg ggacagattg aaaaaatctt caaagtagtt tatccggtcg acgatcatca
     1561 ttttaaagta attctgcact atgggacact cgttatcgat ggagtcacgc cgaatatgat
     1621 agactacttc ggtcgcccgt acgaaggaat cgcggttttc gatggaaaaa aaatcacagt
     1681 aacgggcaca ttgtggaacg ggaataaaat catagacgaa cgcctcatta accctgatgg
     1741 atctttactg ttccgcgtca caattaatgg cgttacaggt tggcgactgt gtgaacgtat
     1801 tctcgcaact agtggtggaa gtgggggcag ccttccagaa acaggaggac accaccacca
     1861 ccaccactaa ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     1921 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1981 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     2041 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2101 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2161 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2221 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2281 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2341 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2401 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2461 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     2521 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2581 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2641 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2701 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2761 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2821 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2881 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2941 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     3001 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     3061 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     3121 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     3181 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     3241 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     3301 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     3361 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     3421 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     3481 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3541 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3601 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3661 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3721 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3781 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3841 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3901 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3961 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4021 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4081 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4141 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4201 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4261 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     4321 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     4381 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     4441 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4501 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4561 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     4621 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4681 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4741 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     4801 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4861 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4921 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4981 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     5041 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     5101 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     5161 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     5221 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     5281 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     5341 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     5401 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     5461 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     5521 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5581 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     5641 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     5701 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     5761 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     5821 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5881 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5941 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     6001 tgattgcgag tgagatattt atgccagcca gccagacgc
//
