LOCUS       sequence-392926-        5952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  superfolder GFP with D134TAG and N150TAA mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5952)
  AUTHORS   Bednar RM, Jana S, Kuppa S, Franklin R, Beckman J, Antony E, Cooley 
            RB, Mehl RA
  TITLE     Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino
            Acids That Enable Efficient Protein Dual-Labeling in Cells.
  JOURNAL   ACS Chem Biol. 2021 Nov 19;16(11):2612-2622. doi: 
            10.1021/acschembio.1c00649. Epub 2021 Sep 30.
  PUBMED    34590824
REFERENCE   2  (bases 1 to 5952)
  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/acschembio.1c00649"; journalName: "ACS Chem Biol"; date: 
            "2021-11-19- 19"; volume: "16"; issue: "11"; pages: "2612-2622"
COMMENT     Sequence Label: pET28-sfGFP[D134TAG+N150TAA]
FEATURES             Location/Qualifiers
     source          1..5952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      33..488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(120..139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     330..351
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(580..1395)
                     /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(580..1395)
                     /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     1303..1322
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1387..1406
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1517..2105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2006..2025
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2259..2276
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2291..2430
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2356..2375)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2516..2538
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2532..2723)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3532..4614)
                     /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     4576..4595
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4615..4692)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4898..4917
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5001..5020
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5001..5019
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5020..5044
                     /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             5059..5081
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5817..5834
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5887..5905)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5901..5948
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
       61 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
      121 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
      181 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
      241 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
      301 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
      361 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
      421 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
      481 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
      541 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
      601 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
      661 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
      721 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
      781 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
      841 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
      901 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
      961 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     1021 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     1081 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     1141 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     1201 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     1261 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     1321 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     1381 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     1441 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1501 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1561 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1621 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1681 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1741 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1801 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1861 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1921 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1981 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2041 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2101 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2161 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2221 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2281 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2341 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     2401 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     2461 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2521 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     2581 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     2641 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     2701 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     2761 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     2821 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     2881 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     2941 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     3001 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     3061 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     3121 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     3181 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     3241 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     3301 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     3361 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     3421 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     3481 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     3541 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3601 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     3661 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     3721 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     3781 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     3841 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     3901 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     3961 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     4021 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     4081 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     4141 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     4201 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     4261 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     4321 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     4381 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     4441 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     4501 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     4561 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     4621 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     4681 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     4741 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     4801 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     4861 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     4921 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     4981 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     5041 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg gttagcaaag
     5101 gtgaagaact gtttaccggc gttgtgccga ttctggtgga actggatggt gatgtgaatg
     5161 gccataaatt tagcgttcgt ggcgaaggcg aaggtgatgc gaccaacggt aaactgaccc
     5221 tgaaatttat ttgcaccacc ggtaaactgc cggttccgtg gccgaccctg gtgaccaccc
     5281 tgacctatgg cgttcagtgc tttagccgct atccggatca tatgaaacgc catgatttct
     5341 ttaaaagcgc gatgccggaa ggctatgtgc aggaacgtac cattagcttc aaagatgatg
     5401 gcacctataa aacccgtgcg gaagttaaat ttgaaggcga taccctggtg aaccgcattg
     5461 aactgaaagg tattgatttt aaagaatagg gcaacattct gggtcataaa ctggaatata
     5521 atttcaacag ccattaagtg tatattaccg ccgataaaca gaaaaatggc atcaaagcga
     5581 actttaaaat ccgtcacaac gtggaagatg gtagcgtgca gctggcggat cattatcagc
     5641 agaatacccc gattggtgat ggcccggtgc tgctgccgga taatcattat ctgagcaccc
     5701 agagcgttct gagcaaagat ccgaatgaaa aacgtgatca tatggtgctg ctggaatttg
     5761 ttaccgccgc gggcattacc cacggtatgg atgaactgta taaaggcagc ctcgagcacc
     5821 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     5881 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     5941 gttttttgct ga
//
