LOCUS       sequence-219913-        5593 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GST-GFP-DHFR; construct generated for pull-down and 
            localization experiments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5593)
  AUTHORS   Caldwell RM, Bermudez JG, Thai D, Aonbangkhen C, Schuster BS, 
            Courtney T, Deiters A, Hammer DA, Chenoweth DM, Good MC
  TITLE     Optochemical Control of Protein Localization and Activity within 
            Cell-like Compartments.
  JOURNAL   Biochemistry. 2018 May 8;57(18):2590-2596. doi: 
            10.1021/acs.biochem.8b00131. Epub 2018 Apr 19.
  PUBMED    29671583
REFERENCE   2  (bases 1 to 5593)
  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: 
            "10.1021/acs.biochem.8b00131"; journalName: "Biochemistry"; date: 
            "2018-05-8- 8"; volume: "57"; issue: "18"; pages: "2590-2596"
COMMENT     Sequence Label: pJB02
FEATURES             Location/Qualifiers
     source          1..5593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     148..167
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        148..166
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             198..220
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             243..893
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP"
     primer_bind     854..876
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             930..950
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(960..962,963..965,966..1676)
                     /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(1005..1026)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1266..1285)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1613..1634
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1716..2192
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             2206..2223
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2241..2258
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2311..2329)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2325..2372
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2409..2864
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2496..2515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2706..2727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2890..2994
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2995..3063,3064..3855)
                     /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(3213..3232)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4026..4614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4515..4534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4768..4785
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4800..4939
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4865..4884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5025..5047
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5041..5229)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="TKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHAD
                     ELYRSCLARFGDDGENL"
ORIGIN
        1 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
       61 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg
      121 ctgcccgaga tctcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt
      181 ttccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg aaacaagatc
      241 tgatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc actcgacttc
      301 ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat gaaggtgata
      361 aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct tattatattg
      421 atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct gacaagcaca
      481 acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa ggagcggttt
      541 tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa actctcaaag
      601 ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt ttatgtcata
      661 aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat gacgctcttg
      721 atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta gtttgtttta
      781 aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc aagtatatag
      841 catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct ccaactagtg
      901 gatctggtgg tggtggcggt tccatgagcg agaatcttta ttttcaggga tccccgcgga
      961 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1021 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1081 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1141 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1201 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1261 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1321 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1381 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     1441 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     1501 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1561 acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1621 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagctcg
     1681 aggacgagct gtataaggcc ggcctggagt ctagaatcag tctgattgcg gcgttagcgg
     1741 tagatcgcgt tatcggcatg gaaaacgcca tgccgtggaa cctgcctgcc gatctcgcct
     1801 ggtttaaacg caacacctta aataaacccg tgattatggg ccgccatacc tgggaatcaa
     1861 tcggtcgtcc gttgccagga cgcaaaaata ttatcctcag cagtcaaccg ggtacggacg
     1921 atcgcgtaac gtgggtgaag tcggtggatg aagccatcgc ggcgtgtggt gacgtaccag
     1981 aaatcatggt gattggcggc ggtcgcgttt atgaacagtt cttgccaaaa gcgcaaaaac
     2041 tgtatctgac gcatatcgac gcagaagtgg aaggcgacac ccatttcccg gattacgagc
     2101 cggatgactg ggaatcggta ttcagcgaat tccacgatgc tgatgcgcag aactctcaca
     2161 gctattgctt tgagattctg gagcggcggt aagcggccgc tctgccacca tcaccaccac
     2221 cactaggggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     2281 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     2341 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     2401 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2461 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2521 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2581 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2641 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2701 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2761 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2821 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     2881 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2941 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     3001 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     3061 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3121 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     3181 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     3241 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     3301 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     3361 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     3421 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     3481 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     3541 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     3601 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     3661 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     3721 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3781 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3841 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3901 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3961 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4021 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4081 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4141 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4201 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttactagtg
     4261 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4321 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4381 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4441 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4501 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4561 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4621 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4681 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4741 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4801 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     4861 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     4921 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4981 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5041 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     5101 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     5161 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5221 ctgtttggtt actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     5281 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     5341 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     5401 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     5461 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     5521 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     5581 gcagcagcag tcg
//
