LOCUS       sequence-310516-        6278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally His-tagged E. coli GlpG 
            C104V/D243G/L244G/F245G/M247C/S248G/M249G/A250G variant (residues 
            87-276) from pET15b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6278)
  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 6278)
  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: 
            pET15-His-∆NGlpG(C104V/D243G/L244G/F245G/M247C/S248G/M249G/A250G)
FEATURES             Location/Qualifiers
     source          1..6278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(909..926)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(936..953)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             972..994
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1009..1033
                     /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     complement(1033..1052)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1034..1052)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1136..1155)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1361..1438
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1439..2521
                     /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(1458..1477)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3833..4024
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4018..4040)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4126..4265
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4181..4200
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4280..4297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4451..5039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4531..4550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5210..6001,6002..6070))
                     /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     5833..5852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6071..6175)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6243..6261
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atcctcgagt tattttcgtt ttcgcgcatt gagcgaatca
      361 acaaaagcca tcgctaaacc cacggctaac ccggcgatgt gtgctccgtt cccgcctcca
      421 cagccacctc caccaaacca tccggcgaca atccagatca gcgcaaagat aattaaccca
      481 cgttgcaggt aaatgccact ttgcggatcg cgttcgccac gtagccagac gtagcccatc
      541 agcgcataca ccacgccaga aagcccgcca aaccacggcc cgctgaattt ttgctgcaca
      601 tagccgctta acagggcgct gataagcgta atgacaatta gcttaccgct accgaggcgt
      661 ttttccaccg caccgccgag ataccaccac cagagcaggt taaagaggat atgcatcagc
      721 gagaagtgca ttaacgcgtg ggtgaagtaa cgccagaact caaatttcag tgttggatcg
      781 aatggccagg ccagccataa catcacttcc tgatcgccga gaatttgcat ggcaataaac
      841 accaccacgg cggcgatcat catcacccag gttaccggac ctgcgcgttc acgcaaggcg
      901 gccatatggc tgccgcgcgg caccaggccg ctgctgtgat gatgatgatg atggctgctg
      961 cccatggtat atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc
     1021 cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc
     1081 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
     1141 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
     1201 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
     1261 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
     1321 ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa
     1381 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     1441 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     1501 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     1561 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     1621 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     1681 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     1741 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     1801 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     1861 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     1921 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     1981 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     2041 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     2101 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     2161 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     2221 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     2281 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     2341 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     2401 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     2461 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     2521 agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc
     2581 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     2641 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     2701 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg
     2761 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac
     2821 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cgacgcgctg ggctacgtct
     2881 tgctggcgtt cgcgacgcga ggctggatgg ccttccccat tatgattctt ctcgcttccg
     2941 gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat gacgaccatc
     3001 agggacagct tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc attggaccgc
     3061 tgatcgtcac ggcgatttat gccgcctcgg cgagcacatg gaacgggttg gcatggattg
     3121 taggcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg
     3181 ccacctcgac ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat
     3241 tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat
     3301 atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc
     3361 ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg
     3421 ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg
     3481 ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta
     3541 aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg
     3601 atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc
     3661 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg
     3721 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt
     3781 catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa
     3841 acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct
     3901 ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga
     3961 ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     4021 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     4081 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     4141 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     4201 ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     4261 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4321 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4381 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4441 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4501 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4561 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4621 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4681 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4741 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4801 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4861 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4921 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4981 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5041 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5101 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5161 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5221 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5281 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5341 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5401 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5461 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5521 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5581 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5641 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5701 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5761 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5821 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5881 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5941 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6001 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6061 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6121 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6181 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     6241 aaaataggcg tatcacgagg ccctttcgtc ttcaagaa
//
