LOCUS       sequence-319729-        4783 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 4783)
  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 4783)
  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: pCGFP-EU
FEATURES             Location/Qualifiers
     source          1..4783
                     /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             579..596
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(609..611,612..1322)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(651..672)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(912..931)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1259..1280
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1338..1361
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     polyA_signal    1511..1632
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1548..1567)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1602..1621
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1639..2094)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1776..1797)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1988..2007
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2121..2225
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2227..2584
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2227..2418
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2250..2270)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2435..2570
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2619..3413
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2619..3413
                     /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
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2673..2692)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3283..3302
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3601..3620)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3645..3692
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4021..4609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4510..4529
                     /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 gctagcgcga
      541 gctctcgaga agcttcgaat tctgcagtcg acgcaggact ggtgccgcgc ggctctgcgg
      601 ccgccgcggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      661 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
      721 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
      781 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
      841 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
      901 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
      961 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1021 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1081 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1141 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1201 accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     1261 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1321 agtccggact cagatctcac catcatcatc accaccatca ctgataggta ccgcgggccc
     1381 gggatccacc ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     1441 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     1501 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1561 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     1621 caatgtatct taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     1681 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     1741 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     1801 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     1861 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     1921 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     1981 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2041 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     2101 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2161 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2221 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     2281 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     2341 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2401 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     2461 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     2521 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     2581 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     2641 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2701 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2761 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     2821 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     2881 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2941 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3001 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3061 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3121 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     3181 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3241 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3301 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3361 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     3421 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     3481 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     3541 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     3601 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     3661 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     3721 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     3781 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     3841 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     3901 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3961 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4021 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4081 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4141 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4201 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4261 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4321 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4381 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4441 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4501 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4561 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4621 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4681 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     4741 caattacggg gtcattagtt catagcccat atatggagtt ccg
//
