LOCUS       sequence-216912-        3589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression and purification of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3589)
  AUTHORS   Busch DJ, Houser JR, Hayden CC, Sherman MB, Lafer EM, Stachowiak JC
  TITLE     Intrinsically disordered proteins drive membrane curvature.
  JOURNAL   Nat Commun. 2015 Jul 24;6:7875. doi: 10.1038/ncomms8875.
  PUBMED    26204806
REFERENCE   2  (bases 1 to 3589)
  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: "Nat 
            Commun."; date: "2015-07-24"; volume: "6:7875. doi"; pages: " 
            10.1038/ncomms8875"
COMMENT     Sequence Label: pRSET his-eGFP
FEATURES             Location/Qualifiers
     source          1..3589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     78..97
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        78..96
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             128..150
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             170..187
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             191..223
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(227..235,236..250)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(266..268,269..271,272..985)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(311..332)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(572..591)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     919..940
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1048..1066)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1062..1109
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1206..1661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1293..1312)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1503..1524
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1687..1791
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1792..1860,1861..2652)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPKERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2010..2029)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2823..3411
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3312..3331
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3565..3582
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagga
       61 tctcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag
      121 aaataatttt gtttaacttt aagaaggaga tatacatatg cggggttctc atcatcatca
      181 tcatcatggt atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga
      241 tgacgataag gatcgatggg gatccatggt gagcaagggc gaggagctgt tcaccggggt
      301 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      361 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      421 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
      481 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      541 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      601 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
      661 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
      721 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
      781 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
      841 ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc
      901 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
      961 cggcatggac gagctgtaca agtaagaatt cgaagcttga tccggctgct aacaaagccc
     1021 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1081 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatctggcg
     1141 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     1201 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1261 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1321 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1381 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1441 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1501 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1561 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1621 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     1681 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1741 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     1801 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     1861 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     1921 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc caaagaacgt
     1981 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     2041 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2101 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2161 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2221 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2281 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     2341 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2401 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2461 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2521 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2581 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2641 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     2701 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     2761 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2821 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2881 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2941 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     3001 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3061 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3121 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3181 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3241 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3301 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3361 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3421 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3481 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3541 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgg
//
