LOCUS       sequence-469209-        5661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid containing GST-tagged EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5661)
  AUTHORS   Addgene Research Program
  TITLE     Addgene GST Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5661)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAG GST EGFP
FEATURES             Location/Qualifiers
     source          1..5661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..111
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(112..180,181..972)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(330..349)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1143..1731
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1632..1651
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1885..1902
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1975..2052
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             2053..3135
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVRRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2072..2091)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    3148..3169
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3184..3189,3190..3207,3208..3214)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3222..3238
                     /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)."
     primer_bind     3246..3262
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3246..3262
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3258..3785
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(3278..3295)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3278..3294)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3287..3309)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(3887..3892,3893..3908,3909..3915)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3923..3939
                     /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)."
     CDS             3962..4615
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     4573..4595
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             4622..4639
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(4640..4642,4643..4645,4646..5356)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4640..4642,4643..4645,4646..5356)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4685..4706)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4946..4965)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5293..5314
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5415..5437)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5582..5600)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
       61 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
      121 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
      181 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
      241 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
      301 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
      361 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
      421 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
      481 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
      541 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
      601 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
      661 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
      721 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
      781 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
      841 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
      901 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
      961 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     1021 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     1081 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1141 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     1201 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     1261 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     1321 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1381 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1441 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1501 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     1561 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     1621 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     1681 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     1741 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     1801 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     1861 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     1921 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc
     1981 atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     2041 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     2101 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     2161 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     2221 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     2281 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     2341 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     2401 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     2461 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     2521 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     2581 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     2641 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     2701 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     2761 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     2821 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     2881 gaagacagct catgttatat cccgccgtca accaccatca aacaggattt tcgcctgctg
     2941 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     3001 cagctgttgc ccgtctcact ggtgagaaga aaaaccaccc tggcgcccaa tacgcaaacc
     3061 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     3121 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     3181 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     3241 tcacacagga aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc
     3301 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     3361 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     3421 tgaatggcga atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc
     3481 tggagtgcga tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg
     3541 gttacgatgc gcccatctac accaacgtaa cctatcccat tacggtcaat ccgccgtttg
     3601 ttcccacgga gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc
     3661 tacaggaagg ccagacgcga attatttttg atggcgttgg aattagctta tcgactgcac
     3721 ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt
     3781 cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt
     3841 tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca
     3901 tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagtatt
     3961 catgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct
     4021 tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa
     4081 atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga
     4141 tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa
     4201 catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt
     4261 ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt
     4321 tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa
     4381 aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga
     4441 tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa
     4501 aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc
     4561 atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc caaaatcgga
     4621 tctggttccg cgtggatcca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     4681 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     4741 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     4801 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     4861 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     4921 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     4981 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     5041 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     5101 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     5161 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     5221 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     5281 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     5341 ggacgagctg tacaaggaat tcatcgtgac tgactgacga tctgcctcgc gcgtttcggt
     5401 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     5461 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     5521 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt ataattcttg aagacgaaag
     5581 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5641 tcaggtggca cttttcgggg a
//
