LOCUS       sequence-335659-        7083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses protein G fused to 3 copies of 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 7083)
  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 7083)
  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-NL3
FEATURES             Location/Qualifiers
     source          1..7083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     568..587
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(607..684)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     890..909
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     993..1012
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        993..1011
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1012..1036
                     /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             1051..1073
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1086..1109
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2913..2930
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2940..2957
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3010..3028)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3024..3071
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3108..3563
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3195..3214)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3405..3426
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3655..4470)
                     /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(3655..4470)
                     /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     4378..4397
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4462..4481
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4592..5180
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5081..5100
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5334..5351
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5366..5505
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5431..5450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5591..5613
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5607..5798)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6607..7083,1..606))
                     /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 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
       61 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
      121 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
      181 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
      241 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
      301 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
      361 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
      421 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
      481 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
      541 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
      601 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
      661 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
      721 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
      781 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
      841 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
      901 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
      961 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     1021 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
     1081 tgggctggtc ccatccgcag ttcgagaaag gtaccatgac atttaaactg ataatcaacg
     1141 gcaaaacctt aaaaggggag atcacaattg aggcagtcga tgcctaggaa gccgagaaaa
     1201 tctttaaaca atatgctaat gattatggta ttgacggaga atggacgtat gacgatgcga
     1261 caaaaacttt caccgtaact gaggaattca ctagtggtgg aagtgggggc agcatggtct
     1321 tcacgttgga agatttcgtt ggagactggc gccagaccgc tggatataac ctggatcaag
     1381 tgcttgagca agggggcgta agttctttgt ttcaaaacct gggcgtgtct gttacaccga
     1441 ttcagagaat cgtcctgtcc ggtgagaacg gactgaagat cgatatacac gttatcatcc
     1501 cctacgaagg tttgtcgggc gatcaaatgg gccaaatcga gaagattttc aaagttgtct
     1561 accccgttga tgatcaccac tttaaggtga tcctgcacta cggtacactt gtcatagacg
     1621 gagtcacacc taacatgatc gactactttg ggcgcccgta cgagggtatc gcagtattcg
     1681 atggtaaaaa aatcacagta actggaacac tttggaacgg taataaaatc atcgacgaac
     1741 gcttgataaa cccagatggg tcgctgctgt tccgggtcac tataaatggc gtgacgggct
     1801 ggagattatg tgaacggata ttggcggggt ccggtggatc ccatatggtg ttcacattag
     1861 aagatttcgt gggcgattgg agacagactg cgggttacaa cttggatcaa gtgttagaac
     1921 aaggtggtgt ttcatcgtta ttccagaact tgggagtaag tgtaaccccg atacaacgca
     1981 tagtccttag cggtgagaat ggactgaaaa ttgatattca tgtaattata ccgtatgagg
     2041 gattgtcggg cgaccagatg ggtcaaatag aaaagatctt caaagtagtg tatccggtcg
     2101 atgaccacca ctttaaggtg atcttgcact atggaactct ggtaattgac ggtgtgaccc
     2161 ctaatatgat tgattatttc ggccgtccgt acgagggcat cgcagtattt gatggtaaga
     2221 agataaccgt tacgggaaca ttatggaatg ggaataagat aatcgatgag cggttgatta
     2281 atccggatgg gtccttactg tttcgtgtaa cgataaatgg agtgactggt tggcgcttat
     2341 gtgaacgtat tctggccggg gctagcggat cacatatggt ctttaccctg gaggactttg
     2401 taggggactg gcggcagacg gccgggtata acctggacca ggtattagaa cagggaggcg
     2461 tctcttcact tttccagaac cttggggtaa gcgtaacgcc tatccagcgg attgtcctgt
     2521 ctggggagaa cgggttaaag atagacatcc atgtcattat cccctacgaa ggcttgtcag
     2581 gcgatcagat gggccaaatt gagaagatat tcaaggtagt ctatccagtc gacgaccacc
     2641 attttaaggt gatattacac tatggaacgc ttgttatcga tggagttact ccaaatatga
     2701 ttgattattt cggacggccc tacgaaggta tagctgtctt tgacggcaaa aagatcacgg
     2761 tgactggtac gttatggaat ggaaacaaaa ttattgatga gcgcttaatt aaccccgatg
     2821 gctcgctgtt atttcgggtc accattaatg gggtcaccgg ctggcgcttg tgtgagagga
     2881 ttctggcgac tagtggtgga agtgggggca gccatcatca tcatcatcat taactcgagc
     2941 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     3001 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     3061 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     3121 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3181 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3241 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3301 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3361 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3421 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3481 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3541 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     3601 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     3661 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     3721 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     3781 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     3841 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     3901 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     3961 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     4021 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     4081 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     4141 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     4201 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     4261 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     4321 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     4381 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     4441 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     4501 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4561 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4621 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4681 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4741 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4801 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4861 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4921 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4981 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5041 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5101 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5161 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5221 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5281 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5341 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     5401 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5461 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     5521 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5581 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5641 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     5701 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5761 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5821 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5881 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     5941 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     6001 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     6061 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     6121 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     6181 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     6241 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     6301 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     6361 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     6421 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     6481 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     6541 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     6601 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6661 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     6721 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     6781 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     6841 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     6901 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6961 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     7021 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     7081 ttt
//
