LOCUS       sequence-319761-        6260 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 6260)
  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 6260)
  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-BC
FEATURES             Location/Qualifiers
     source          1..6260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..115
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             122..835
                     /codon_start=1
                     /product="GFP variant optimized for excitation by UV light"
                     /label=GFPuv
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(152..180)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     775..795
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             890..907
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             992..1009
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1062..1080)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1076..1123
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1160..1615
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1247..1266)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1457..1478
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1641..1745
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1746..1814,1815..2606)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1964..1983)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2777..3365
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3266..3285
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3519..3536
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3551..3690
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3616..3635)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3776..3798
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3792..3983)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4792..5874)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5836..5855
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5875..5952)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6158..6177
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatc atcacaccgg
      121 tatgagtaaa ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg
      181 tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg
      241 aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttcctt ggcctacact
      301 tgtcactact ttctcttatg gtgttcaatg cttttcccgt tatccggatc atatgaaacg
      361 gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaacgca ctatatcttt
      421 caaagatgac gggaactaca agacgcgtgc tgaagtcaag tttgaaggtg atacccttgt
      481 taatcgtatc gagttaaaag gtattgattt taaagaagat ggaaacattc tcggacacaa
      541 actcgaatac aactataact cacacaatgt atacatcacg gcagacaaac aaaagaatgg
      601 aatcaaagct aacttcaaaa ttcgccacaa cattgaagat gggtccgttc aactagcaga
      661 ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta
      721 cctgtccaca caatctaaac tttcgaaaga tcccaacgaa aagcgtgacc acatggtcct
      781 tcttgagttt gtaactgctg ctgggattac acatggcatg gatgaactct acaaatcgag
      841 ctcgaacaac aacaacaata acaataacaa caacctcggg actagtggcc tggtgcctag
      901 gggcagcgcc atggatatcg gaattaattc ggatccgaat tcgccgtcga caagcttgcg
      961 gccgcactcg agtgatagct gattaatcga gcaccaccac caccaccact gagatccggc
     1021 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1081 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1141 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1201 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1261 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1321 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1381 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1441 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1501 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1561 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     1621 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1681 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1741 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1801 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1861 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1921 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     1981 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2041 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2101 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2161 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2221 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2281 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2341 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2401 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     2461 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2521 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2581 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2641 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2701 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2761 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2821 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2881 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2941 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3001 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3061 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3121 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3181 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3241 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3301 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3361 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3421 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3481 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3541 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3601 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3661 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3721 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3781 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     3841 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     3901 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3961 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4021 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4081 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4141 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4201 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4261 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4321 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     4381 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4441 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4501 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4561 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4621 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4681 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4741 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4801 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4861 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     4921 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     4981 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5041 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     5101 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5161 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5221 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5281 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5341 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5401 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5461 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5521 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5581 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5641 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5701 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5761 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     5821 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     5881 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     5941 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     6001 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     6061 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     6121 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     6181 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     6241 agatctcgat cccgcgaaat
//
