LOCUS       sequence-335361-        6696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses protein G fused to mNeonGreen and 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 6696)
  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 6696)
  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-mNG-NL
FEATURES             Location/Qualifiers
     source          1..6696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     914..933
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(953..1030)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1236..1255
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1339..1358
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1339..1357
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1358..1382
                     /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             1397..1419
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1432..1455
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2872..2889
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2899..2916
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2969..2987)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2983..3030
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3067..3522
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3154..3173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3364..3385
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3614..4429)
                     /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(3614..4429)
                     /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     4337..4356
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4421..4440
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4551..5139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5040..5059
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5293..5310
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5325..5464
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5390..5409)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5550..5572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5566..5757)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6566..6696,1..952))
                     /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 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
       61 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
      121 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
      181 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
      241 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
      301 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
      361 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
      421 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
      481 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
      541 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
      601 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
      661 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
      721 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
      781 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
      841 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
      901 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
      961 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     1021 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     1081 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     1141 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     1201 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     1261 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     1321 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1381 cccctctaga aataattttg tttaacttta agaaggagat ataccatggg ctggtcccat
     1441 ccgcagttcg agaaaggtac catgacattt aaactgataa tcaacggcaa aaccttaaaa
     1501 ggggagatca caattgaggc agtcgatgcc taggaagccg agaaaatctt taaacaatat
     1561 gctaatgatt atggtattga cggagaatgg acgtatgacg atgcgacaaa aactttcacc
     1621 gtaactgagg aattctcagg cagccatatg gtaagtaaag gtgaagaaga caatatggct
     1681 tctctgcctg ccacacatga gcttcatatt tttgggagca taaacggagt ggatttcgac
     1741 atggtaggtc agggtacggg gaaccctaac gatggatatg aggagttgaa tcttaaaagc
     1801 acaaagggtg atctgcagtt ctcgccctgg atcctggtgc cgcatatagg ttatggtttc
     1861 catcagtatc ttccataccc ggatggcatg agcccttttc aggccgcaat ggtagatggc
     1921 tcaggatatc aagtgcatcg gaccatgcag tttgaagatg gggcgtcttt gacggtaaat
     1981 tacaggtaca cctatgaggg tagccatata aagggagaag cgcaggtgaa gggaactgga
     2041 ttcccagcgg atggcccagt catgacaaac agcctcaccg ctgctgattg gtgccgatcc
     2101 aagaaaacgt atccaaacga taaaactatc atttctactt ttaagtggtc ctatacaaca
     2161 ggaaacggga aacgctatcg ttcaacggcc cgcacgacct acacgtttgc aaagccaatg
     2221 gctgcgaatt atctgaaaaa ccagccgatg tatgtgttcc gtaaaaccga actgaaacat
     2281 tctaaaacgg agctcaattt caaggaatgg cagaaggcat ttaccggttt tgaagatttc
     2341 gtgggtgatt ggcgacaaac ggccggttac aatttggatc aggtgttaga acaagggggc
     2401 gtaagctccc tgttccagaa tttaggagtg agcgtgacac ctattcagcg cattgtgctg
     2461 agcggcgaaa atggtctgaa aattgatatt catgtgatca tcccttacga aggcctgtct
     2521 ggggatcaaa tgggacagat tgaaaaaatc ttcaaagtag tttatccggt cgacgatcat
     2581 cattttaaag taattctgca ctatgggaca ctcgttatcg atggagtcac gccgaatatg
     2641 atagactact tcggtcgccc gtacgaagga atcgcggttt tcgatggaaa aaaaatcaca
     2701 gtaacgggca cattgtggaa cgggaataaa atcatagacg aacgcctcat taaccctgat
     2761 ggatctttac tgttccgcgt cacaattaat ggcgttacag gttggcgact gtgtgaacgt
     2821 attctcgcaa ctagtggtgg aagtgggggc agccttccag aaacaggagg acaccaccac
     2881 caccaccact aactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     2941 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     3001 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     3061 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3121 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3181 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3241 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3301 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3361 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3421 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3481 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     3541 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3601 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     3661 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3721 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     3781 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     3841 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     3901 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     3961 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     4021 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     4081 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     4141 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     4201 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     4261 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     4321 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     4381 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     4441 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     4501 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4561 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4621 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4681 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4741 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4801 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4861 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4921 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4981 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5041 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5101 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5161 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5221 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5281 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     5341 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     5401 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5461 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5521 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5581 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5641 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5701 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5761 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5821 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5881 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5941 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     6001 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     6061 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     6121 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     6181 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     6241 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     6301 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     6361 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     6421 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     6481 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6541 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6601 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     6661 gggtggtttt tcttttcacc agtgagacgg gcaaca
//
