LOCUS       sequence-310752-        5538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of rhomboid protease catalytic mutant 
            HiGlpG-S116A,H196A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5538)
  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 5538)
  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-HiGlpG- S116A, H196A
FEATURES             Location/Qualifiers
     source          1..5538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             75..602
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(95..112)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(95..111)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(104..126)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(704..709,710..725,726..732)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    740..756
                     /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             779..1432
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1390..1412
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1439..1462
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2109..2131)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2276..2294)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2362..2466
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2467..2535,2536..3327)
                     /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(2685..2704)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3498..4086
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3987..4006
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4240..4257
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4330..4407
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4408..5490
                     /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(4427..4446)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta caacgtcgtg
      121 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
      181 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      241 atggcgaatg gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg
      301 agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt
      361 acgatgcgcc catctacacc aacgtgacct atcccattac ggtcaatccg ccgtttgttc
      421 ccacggagaa tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac
      481 aggaaggcca gacgcgaatt atttttgatg gcgttggaat tagcttatcg actgcacggt
      541 gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt
      601 aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc
      661 gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg
      721 gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagtattcat
      781 gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt
      841 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg
      901 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg
      961 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat
     1021 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga
     1081 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga
     1141 ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac
     1201 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt
     1261 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa
     1321 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg
     1381 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct
     1441 ggaagttctg ttccaggggc ccctgggatc caaaaacttt ttagctcaac aaggaaaaat
     1501 cacgctaatt ctcaccgcac tttgtgtgct tatttatctt gctcaacagc tgggttttga
     1561 agacgatatt atgtatttga tgcattaccc cgcttacgaa gagcaagata gtgaagtgtg
     1621 gcgttatatc tcacatacat tggtacattt atctaatttg catattttgt ttaatctgtc
     1681 ttggtttttt attttcggtg gaatgattga gcgcactttt ggctcggtta aattattgat
     1741 gttgtatgtt gtagcatctg ccataacagg atatgtgcaa aattatgtgt ctggccccgc
     1801 tttctttggg cttgctggtg ttgtatatgc ggttctaggt tatgtattta tacgtgataa
     1861 attgaatcat catttatttg atctgccaga aggttttttt acaatgttat tggtggggat
     1921 cgcattaggt tttattagcc ccttatttgg tgttgaaatg ggaaatgctg cagctatttc
     1981 aggcttgatt gtcggcttga tttggggatt tattgatagc aaattgcgca aaaattcgct
     2041 agagtaagcc ggccgcatcg tgactgactg acgatctgcc tcgcgcgttt cggtgatgac
     2101 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     2161 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     2221 gccatgaccc agtcacgtag cgatagcgga gtgtataatt cttgaagacg aaagggcctc
     2281 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     2341 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2401 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2461 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2521 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2581 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2641 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2701 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2761 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2821 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2881 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2941 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3001 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3061 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3121 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3181 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3241 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3301 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3361 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3421 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3481 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3541 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3601 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3661 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3721 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3781 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3841 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3901 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3961 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4021 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4081 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4141 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4201 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4261 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4321 ataaattccg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     4381 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     4441 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     4501 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     4561 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     4621 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     4681 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     4741 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     4801 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     4861 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     4921 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     4981 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     5041 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     5101 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     5161 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     5221 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     5281 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     5341 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac cacgctagcg
     5401 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     5461 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     5521 tcattaggca ccccaggc
//
