LOCUS       sequence-310495-        5840 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally GFP- and HA-tagged E. coli GlpG 
            D243G/L244G/F245G/M247G/S248G/M249G/A250G variant from pGEX 4P-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5840)
  AUTHORS   Cho S, Baker RP, Ji M, Urban S
  TITLE     Ten catalytic snapshots of rhomboid intramembrane proteolysis from 
            gate opening to peptide release.
  JOURNAL   Nat Struct Mol Biol. 2019 Oct;26(10):910-918. doi: 
            10.1038/s41594-019-0296-9. Epub 2019 Sep 30.
  PUBMED    31570873
REFERENCE   2  (bases 1 to 5840)
  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: 
            "10.1038/s41594-019-0296-9"; journalName: "Nat Struct Mol Biol"; 
            date: "2019-10"; volume: "26"; issue: "10"; pages: "910-918"
COMMENT     Sequence Label: 
            pGEX-HA-GlpG(D243G/L244G/F245G/M247G/S248G/M249G/A250G)
FEATURES             Location/Qualifiers
     source          1..5840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1890..1912)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2057..2075)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2143..2247
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2248..2316,2317..3108)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2466..2485)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3279..3867
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3768..3787
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4021..4038
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4111..4188
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4189..5271
                     /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     complement(4208..4227)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5320..5325,5326..5343,5344..5350)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5358..5374
                     /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     5382..5398
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5382..5398
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5394..5840,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5414..5431)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5414..5430)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5423..5445)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc ccggaattcc
      961 cgggtcgact cgagatgtac ccatacgatg ttcctgacta tttgatgatt acctcttttg
     1021 ctaacccccg cgtggcgcag gcgtttgttg attacatggc gacgcagggt gttatcctca
     1081 cgattcaaca acataaccaa agcgatgtct ggctggcgga tgagtcccag gccgagcgcg
     1141 tacgggcgga gctggcgcgt tttctcgaaa acccggcaga tccgcgttat ctggcggcga
     1201 gctggcaggc aggccatacc ggcagtggcc tgcattatcg ccgttatcct ttctttgccg
     1261 ccttgcgtga acgcgcaggt ccggtaacct gggtgatgat gatcgcctgc gtggtggtgt
     1321 ttattgccat gcaaattctc ggcgatcagg aagtgatgtt atggctggcc tggccattcg
     1381 atccaacact gaaatttgag ttctggcgtt acttcaccca cgcgttaatg cacttctcgc
     1441 tgatgcatat cctctttaac ctgctctggt ggtggtatct cggcggtgcg gtggaaaaac
     1501 gcctcggtag cggtaagcta attgtcatta cgcttatcag cgccctgtta agcggctatg
     1561 tgcagcaaaa attcagcggg ccgtggtttg gcgggctttc tggcgtggtg tatgcgctga
     1621 tgggctacgt ctggctacgt ggcgaacgcg atccgcaaag tggcatttac ctgcaacgtg
     1681 ggttaattat ctttgcgctg atctggattg tcgccggatg gtttggtgga ggtggcggtg
     1741 gaggcgggaa cggagcacac atcgccgggt tagccgtggg tttagcgatg gcttttgttg
     1801 attcgctcaa tgcgcgaaaa cgaaaataag cggccgcatc gtgactgact gacgatctgc
     1861 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     1921 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     1981 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat
     2041 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     2101 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2161 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2221 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     2281 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2341 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2401 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2461 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     2521 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2581 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2641 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2701 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2761 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     2821 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2881 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2941 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     3001 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3061 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3121 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     3181 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3241 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3301 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3361 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3421 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3481 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3541 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3601 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3661 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3721 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3781 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3841 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3901 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3961 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4021 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4081 ctgtgcggta tttcacaccg cataaattcc gacaccatcg aatggcgcaa aacctttcgc
     4141 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     4201 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     4261 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     4321 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     4381 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     4441 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     4501 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     4561 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     4621 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     4681 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     4741 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     4801 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     4861 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     4921 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     4981 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     5041 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     5101 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     5161 aaaagaaaaa ccacgctagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     5221 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     5281 aattaatgta agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     5341 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     5401 tgattacgga ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     5461 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     5521 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct
     5581 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg
     5641 atactgtcgt cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca
     5701 acgtgaccta tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt
     5761 gttactcgct cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta
     5821 tttttgatgg cgttggaatt
//
