LOCUS       sequence-319762-        6242 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 6242)
  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 6242)
  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-BC
FEATURES             Location/Qualifiers
     source          1..6242
                     /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             170..187
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             191..901
                     /codon_start=1
                     /product="GFP variant optimized for excitation by UV light"
                     /label=GFPuv
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(218..246)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     841..861
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             902..925
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             935..967
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             974..991
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1044..1062)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1058..1105
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1142..1597
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1229..1248)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1439..1460
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1623..1727
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1728..1796,1797..2588)
                     /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(1946..1965)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2759..3347
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3248..3267
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3501..3518
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3533..3672
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3598..3617)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3758..3780
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3774..3965)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4774..5856)
                     /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     5818..5837
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5857..5934)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6140..6159
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacccat ggccaaggat atcggaatta attcggatcc
      121 gaattcgagc tccgtcgaca agcttgcggc cgcactcgag agtagtggcc tggtgccgcg
      181 cggcagcgtg agtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
      241 agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac
      301 atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg ttccttggcc
      361 tacacttgtc actactttct cttatggtgt tcaatgcttt tcccgttatc cggatcatat
      421 gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat
      481 atctttcaaa gatgacggga actacaagac gcgtgctgaa gtcaagtttg aaggtgatac
      541 ccttgttaat cgtatcgagt taaaaggtat tgattttaaa gaagatggaa acattctcgg
      601 acacaaactc gaatacaact ataactcaca caatgtatac atcacggcag acaaacaaaa
      661 gaatggaatc aaagctaact tcaaaattcg ccacaacatt gaagatgggt ccgttcaact
      721 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa
      781 ccattacctg tccacacaat ctaaactttc gaaagatccc aacgaaaagc gtgaccacat
      841 ggtccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatg aactctacaa
      901 acaccaccac caccaccacc accactgagc tagccagcca gaactcgccc cggaagaccc
      961 cgaggatgtc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     1021 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1081 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1141 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1201 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1261 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1321 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1381 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1441 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     1501 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     1561 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     1621 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1681 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1741 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1801 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1861 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1921 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     1981 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2041 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     2101 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     2161 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     2221 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     2281 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2341 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2401 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2461 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2521 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2581 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2641 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2701 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2761 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2821 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2881 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2941 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3001 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3061 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3121 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3181 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3241 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3301 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3361 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3421 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3481 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3541 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3601 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3661 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3721 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3781 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3841 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3901 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3961 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4021 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4081 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4141 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4201 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4261 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4321 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4381 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4441 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4501 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4561 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     4621 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4681 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4741 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4801 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4861 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4921 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4981 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5041 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5101 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5161 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5221 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5281 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5341 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5401 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5461 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5521 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5581 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5641 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5701 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5761 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     5821 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     5881 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5941 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     6001 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6061 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     6121 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     6181 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     6241 at
//
