LOCUS       sequence-135934-        7306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GST-EndoE from E. faecalis  in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7306)
  AUTHORS   Collin M, Fischetti VA
  TITLE     A novel secreted endoglycosidase from Enterococcus faecalis with 
            activity on human immunoglobulin G and ribonuclease B.
  JOURNAL   J Biol Chem. 2004 May 21;279(21):22558-70. Epub 2004 Mar 17.
  PUBMED    15028731
REFERENCE   2  (bases 1 to 7306)
  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: "J Biol 
            Chem."; date: "2004-05-21"; volume: "279(21)"; pages: "22558-70. 
            Epub 2004 Mar 17"
COMMENT     Sequence Label: pGEXndoE
FEATURES             Location/Qualifiers
     source          1..7306
                     /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             921..932
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3356..3378)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3523..3541)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3609..3713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3714..3782,3783..4574)
                     /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(3932..3951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4745..5333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5234..5253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5487..5504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5577..5654
                     /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             5655..6737
                     /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(5674..5693)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    6750..6771
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6786..6791,6792..6809,6810..6816)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6824..6840
                     /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     6848..6864
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6848..6864
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6860..7306,1..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(6880..6897)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6880..6896)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6889..6911)
                     /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 atcgaaggtc gtgggatccc caatgagccg acccaagaga
      961 aacattttat ggtttattat cgggcttggc gtgacaaaac gatgcaagga gttaatacaa
     1021 cattgccaga tgaaaattgg ctaacgatgc acgatattcc ttatggtatt gacattgtga
     1081 atgtctttag ttatgtgcca aaaggacaag aagcacttgc acagccattt tatgatacgt
     1141 taaaaaatga gtatgcgcca gcactgcatg cacgaggtgt tcgtttagtt cgagggattg
     1201 attacagcga gctattaaaa gttccttatg caggaacaac gcctacagaa gcagaatttg
     1261 atgcttatgc gaaagagttg ttaaccaaat ttgtcgatga tttaggaatt gatgggttag
     1321 atattgacat ggaaactcgt ccaagtgaaa aagatattgt gctatctaat ggtgtcattc
     1381 gtgcattatc aaaatacatt ggaccgaagt cgggaacgga tcgtccattt ttgtatgata
     1441 ccaatgcaga atatttacca cctttacaag atgtcagtga ctgtttcgat tttctcgcgt
     1501 atcaacagta tggtagcgat gacaaacgca cgcaacgagc attaaataat ttaagtccgg
     1561 ttctcaatgg ggaacgattt gttccaggat taactttccc agaagagcaa gatcgcaacc
     1621 gctggtatga cacaaaagag ccgtatatgg aaagtaacat gtataaagta gctcgttatt
     1681 cttatgaaaa taatttaggg ggcatgttcc tctatgcctt agatcgcgat ggtcgcacct
     1741 ataatgaaga cgatttaaat cagataaaac cttctaattt actttggaca aaaacggcca
     1801 ttgcagagag taaaggcgtt tctcttgcag agatgaaagc ggctgcgcaa cactatttaa
     1861 aacgaattag ctacgccaac acagaccttg aagcacaaaa taaagccgca gaagcagtga
     1921 cacaagcaac aacgctttat gatgtgaata aagctatttt aggtggcgat tatggccaag
     1981 ggctttcaaa tacctatgat gctgaattag aaaaaggcct gttagccatt gacttaacca
     2041 ctttatatcg tgcgttggat caagcagttg cagccattga aaaggcagaa agctatacac
     2101 cagaaacgat tcaagcacta caaacaacaa aagagtcagt cgcaacagaa cttgcgggaa
     2161 aaacgtatac agccgcgcaa gtgactactt ggcaaacgga ggtccaaaca gctttggata
     2221 atttaaaaga gaaacaaaca caacctttaa aaagtgtctt ctccattgat gcgggaagaa
     2281 aatatttttc cgtggaacaa ctggaagaat tagtggccaa agccagtcaa aatgggtaca
     2341 cagatgtcca attaatttta ggaaatgatg gcttacggtt tattttggat gatatgtcgg
     2401 tcaatgtgaa tggtaaaaaa tacaaccaca accgggtttc aaaagcaatc caacgaggca
     2461 acaacgcata ttacaatgat cctaatggca acgcgttaac acaaaaagaa atggatcggt
     2521 tgttggcttt tgcgaaagcg cgcaacatca acatcattcc cgtgattaac agtccaggac
     2581 atatggatgc gttgttagtg gccatggaaa aactagcgat taaaaatccc gcttttgatg
     2641 gctcaaaacg aacagtagat ttagggaacc aaaaggcagt gaatttcaca aaggcgatta
     2701 tcagtaagta cgtggcttat ttttccgcgc atagtgaaat tttcaatttt ggtggcgatg
     2761 agtatgcaaa tgatgtcgac acaggcggtt gggcgaaact gcaatcttct gggcgctaca
     2821 aggattttgt cgcttatgcg aatgatttag ctaaaataat taaagatgcg ggcatgcagc
     2881 caatgagctt taatgatggc atttattaca acagcgacga ttctttcggt acatttgacc
     2941 cagagattat tatttcttat tggacagccg gttggagtgg gtatgacgta gccaaacctg
     3001 agtactttgt tcaaaaagga cacaaaattt ttaataccaa tgatgcgtgg tattgggtcg
     3061 ctggcaatgt tgattctggc atttatcaat atgacgatgc tttagcaaat atgtcgaaaa
     3121 aagcatttac agatgtgcca gcaggtagcc caaatcttcc aattattgga agtattcaat
     3181 gtgtttggta tgatgaccct cgtcgtgact atgattttga acgaatttat acgctaatgg
     3241 atacgttctc ggaaaattat cgtgagtata tggtggttaa aaatcactag ctcgagcggc
     3301 cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3361 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3421 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3481 cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat
     3541 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     3601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     3961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     4261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4861 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     4921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5521 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata aattccgaca
     5581 ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     5641 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     5701 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     5761 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     5821 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     5881 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     5941 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     6001 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     6061 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     6121 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     6181 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     6241 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     6301 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     6361 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     6421 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     6481 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     6541 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     6601 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     6661 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     6721 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     6781 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     6841 tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg
     6901 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     6961 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     7021 cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc cggaaagctg
     7081 gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca
     7141 cggttacgat gcgcccatct acaccaacgt aacctatccc attacggtca atccgccgtt
     7201 tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg atgaaagctg
     7261 gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaatt
//
