LOCUS       sequence-194647-        4148 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses rsEGFP2 in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4148)
  AUTHORS   Grotjohann T, Testa I, Reuss M, Brakemann T, Eggeling C, Hell SW, 
            Jakobs S
  TITLE     rsEGFP2 enables fast RESOLFT nanoscopy of living cells.
  JOURNAL   eLife. 2012 Dec 31:1:e00248. doi: 10.7554/eLife.00248
  PUBMED    23330067
REFERENCE   2  (bases 1 to 4148)
  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: "eLife. 2012
            Dec 31:1:e00248. doi: 10.7554/eLife.00248"
COMMENT     Sequence Label: pQE31-rsEGFP2
FEATURES             Location/Qualifiers
     source          1..4148
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             154..870
                     /codon_start=1
                     /product="reversibly and rapidly switchable enhanced green 
                     fluorescent protein (Grotjohann et al., 2012)"
                     /label=rsEGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLAYGVLCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     SNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(199..220)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(460..479)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     807..828
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      895..989
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1033..1692
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1099..1118)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1757..1843
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1890..1912)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1998..2138
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2053..2072
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2153..2170)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2324..2912)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2404..2423)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3083..3874,3875..3943))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3706..3725
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3944..4048)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4116..4134
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatctcacc atcaccatca ccatacggat ccgatggtga gcaagggcga ggagctgttc
      181 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      241 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      301 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctggc ctacggcgtg
      361 ctgtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      421 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      481 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
      541 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
      601 aacgtctata tcatggccga caagcagaag aacggcatca agtctaactt caagatccgc
      661 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
      721 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc
      781 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
      841 atcactctcg gcatggacga gctgtacaag taaaagctta attagctgag cttggactcc
      901 tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt
      961 gccgccgggc gttttttatt ggtgagaatc caagctagct tggcgagatt ttcaggagct
     1021 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
     1081 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
     1141 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
     1201 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt tcgtatggca
     1261 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
     1321 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
     1381 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
     1441 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
     1501 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat
     1561 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
     1621 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
     1681 ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg ggtaatgact
     1741 ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1801 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccct ctagagctgc
     1861 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     1921 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     1981 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     2041 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     2101 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     2161 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2221 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2281 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2341 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2401 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2461 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2521 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2581 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2641 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2701 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2761 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2821 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2881 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2941 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3001 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3061 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3121 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3181 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3241 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3301 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3361 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3421 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3481 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3541 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3601 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3661 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3721 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3781 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3841 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3901 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3961 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4021 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     4081 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     4141 gtcttcac
//
