LOCUS       sequence-310726-        5532 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of rhomboid protease mutant S*HiGlpG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5532)
  AUTHORS   Mihaljevic L, Urban S
  TITLE     Decoding the Functional Evolution of an Intramembrane Protease 
            Superfamily by Statistical Coupling Analysis.
  JOURNAL   Structure. 2020 Aug 10. pii: S0969-2126(20)30279-3. doi: 
            10.1016/j.str.2020.07.015.
  PUBMED    32795403
REFERENCE   2  (bases 1 to 5532)
  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: "Structure. 
            2020 Aug 10. pii: S0969-2126(20)30279-3. doi: 
            10.1016/j.str.2020.07.015."
COMMENT     Sequence Label: pGEX-S*HiGlpG
FEATURES             Location/Qualifiers
     source          1..5532
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1400..1422)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1567..1585)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1653..1757
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1758..1826,1827..2618)
                     /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(1976..1995)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2789..3377
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3278..3297
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3531..3548
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3621..3698
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3699..4781
                     /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(3718..3737)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4830..4835,4836..4853,4854..4860)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4868..4884
                     /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     4892..4908
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4892..4908
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4904..5431
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4924..4941)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4924..4940)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4933..4955)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggccca tgaaaaactt tttagctcaa
      781 caaggaaaaa tcacgctaat tctcaccgca ctttgtgtgc ttatttatct tgctcaacag
      841 ctgggttttg aagacgatat tatgtatttg atgcattacc ccgcttacga agagcaagat
      901 agtgaagtgt ggcgttatat cacacatgca ttggtacatt tatctaattt gcatattttg
      961 tttaatctgt cttggtggtt tattctgggt ggaatgattg agcgcacttt tggctcggtt
     1021 aaattattga tgttgactgt tgtaagcgct gccataagcg gatatgtgca aaattatgtg
     1081 tctggcccct ggttctttgg gctttctggt gttgtatatg cggttctagg ttatgtattt
     1141 atacgtgatg aattgaatca tcatttattt gatctgccag aaggtttttt tacattctta
     1201 ttggtgtgga tcgcattagg ttttattagc cccttatttg gtgttgaaat ggcgaatgct
     1261 gcacatattg caggcttgat tgtcggcttg atttggggat ttattgatag caaattgcgc
     1321 aaaaattcgc tagagtaagc cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt
     1381 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     1441 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     1501 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac
     1561 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1621 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1681 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1741 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1801 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1861 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1921 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1981 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     2041 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2101 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2161 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2221 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2281 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2341 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2401 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2461 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2521 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2581 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2641 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2701 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2761 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2821 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2881 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2941 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3001 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3061 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3121 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3181 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3241 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3301 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3361 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3421 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3481 ccgcctttga gtgagctgat accgctcgcc acagccgaac gaccgagcgc agcgagtcag
     3541 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3601 tttcacaccg cataaattcc gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
     3661 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     3721 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     3781 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     3841 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     3901 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     3961 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     4021 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     4081 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     4141 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     4201 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     4261 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     4321 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     4381 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     4441 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     4501 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     4561 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     4621 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     4681 ccacgctagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4741 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
     4801 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     4861 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga
     4921 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     4981 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     5041 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc
     5101 accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt
     5161 cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta
     5221 tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct
     5281 cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg
     5341 cgttggaatt agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
     5401 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
     5461 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
     5521 tgaaatgagc tg
//
