LOCUS       sequence-207846-        6451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of EGFP with N-terminal lanthanide binding tag 
            and C-terminal His6 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6451)
  AUTHORS   Bhattacharya K, Bernasconi L, Picard D
  TITLE     Luminescence resonance energy transfer between genetically encoded 
            donor and acceptor for protein-protein interaction studies in the 
            molecular chaperone HSP70/HSP90 complexes.
  JOURNAL   Sci Rep. 2018 Feb 12;8(1):2801. doi: 10.1038/s41598-018-21210-6.
  PUBMED    29434293
REFERENCE   2  (bases 1 to 6451)
  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"; journalName: "Sci Rep."; 
            date: "2018-02-12"; volume: "8(1):2801. doi"; pages: " 
            10.1038/s41598-018-21210-6"
COMMENT     Sequence Label: pET/LEGH
FEATURES             Location/Qualifiers
     source          1..6451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     84..103
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        84..102
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    103..127
                     /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             142..164
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(246..248,249..251,252..962)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(246..248,249..251,252..962)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(291..312)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(552..571)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     899..920
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             966..983
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1036..1054)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1050..1097
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1272..1277,1278..1294,1295..1300))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1327..1345)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1413..1517
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1518..1586,1587..2378)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1736..1755)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2549..3137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3038..3057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3291..3308
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3323..3462
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3388..3407)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3548..3570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3564..3755)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6110..6129
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6149..6226)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6432..6451
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
       61 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
      121 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggctata
      181 tagatacaaa caatgatgga tggtatgaag gagatgaact attagcagct ggaccggtcg
      241 ccatcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      301 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
      361 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
      421 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
      481 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
      541 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
      601 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
      661 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
      721 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
      781 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
      841 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
      901 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
      961 agagccacca tcaccatcac cattaggatc cggctgctaa caaagcccga aaggaagctg
     1021 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1081 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg
     1141 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
     1201 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
     1261 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac
     1321 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1381 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1441 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1501 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1561 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1621 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1681 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1741 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     1801 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1861 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1921 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1981 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2041 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2101 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2161 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2221 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2281 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2341 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2401 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2461 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2521 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2581 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2641 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2701 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2761 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2821 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2881 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2941 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3001 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3061 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3121 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3181 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3241 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3301 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3361 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3421 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3481 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3541 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3601 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3661 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3721 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3781 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3841 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3901 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     3961 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4021 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4081 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4141 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4201 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg
     4261 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
     4321 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
     4381 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
     4441 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
     4501 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
     4561 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
     4621 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
     4681 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
     4741 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4801 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4861 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4921 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4981 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     5041 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aactgtcgtg
     5101 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     5161 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     5221 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5281 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5341 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5401 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5461 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5521 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5581 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5641 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5701 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5761 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5821 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5881 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5941 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     6001 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     6061 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     6121 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     6181 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     6241 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     6301 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     6361 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     6421 tcttccccat cggtgatgtc ggcgatatag g
//
