LOCUS       sequence-319758-        4785 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for rapid and efficient membrane protein precrystallization 
            screening strategy utilizing green fluorescent protein and 
            fluorescence-detection size-exclusion chromatography (FSEC)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4785)
  AUTHORS   Kawate T, Gouaux E
  TITLE     Fluorescence-detection size-exclusion chromatography for 
            precrystallization screening of integral membrane proteins.
  JOURNAL   Structure. 2006 Apr;14(4):673-81. doi: 10.1016/j.str.2006.01.013.
  PUBMED    16615909
REFERENCE   2  (bases 1 to 4785)
  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: 
            "10.1016/j.str.2006.01"; journalName: "Structure"; date: "2006-04"; 
            volume: "14"; issue: "4"; pages: "673-81"
COMMENT     Sequence Label: pNGFP-EU
FEATURES             Location/Qualifiers
     source          1..4785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             556..579
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     regulatory      583..592
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(589..591,592..594,595..1305)
                     /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(634..655)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(895..914)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1242..1263
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1324..1341
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     polyA_signal    1513..1634
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1550..1569)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1604..1623
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1641..2096)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1778..1799)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1990..2009
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2123..2227
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2229..2586
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2229..2420
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2252..2272)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2437..2572
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2621..3415
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMQRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2675..2694)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3285..3304
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3603..3622)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3647..3694
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4023..4611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4512..4531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgacg
      541 agctcagcca ccatgcacca tcatcatcac caccatcacg cggccgccat ggtgagcaag
      601 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      661 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
      721 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
      781 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
      841 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
      901 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
      961 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1021 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1081 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1141 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1201 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1261 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg actcagatct
     1321 ggactggtgc cgcgcggctc tcgagaagct tcgaattctg cagtcgacgg taccgcgggc
     1381 ccgggatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     1441 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     1501 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     1561 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     1621 atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     1681 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     1741 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     1801 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     1861 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     1921 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     1981 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     2041 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     2101 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2161 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2221 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     2281 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2341 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2401 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2461 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2521 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     2581 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     2641 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     2701 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     2761 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     2821 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     2881 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     2941 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cagcggctgc atacgcttga
     3001 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3061 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3121 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     3181 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3241 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3301 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3361 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3421 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3481 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3541 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     3601 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     3661 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     3721 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     3781 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     3841 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     3901 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3961 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4021 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4081 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4141 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4201 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4261 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4321 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4381 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4441 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4501 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4561 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4621 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4681 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta
     4741 atcaattacg gggtcattag ttcatagccc atatatggag ttccg
//
