LOCUS       sequence-357121-        6860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TtEnd_E329A_pEHISTEV is a mutant form of TtEnd5A_pEHISTEV vector. It
            expresess mutated TtEnd5A in which Glu329 has been mutated with 
            Ala329.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6860)
  AUTHORS   Hussain N, Mikolajek H, Harrison PJ, Paterson N, Akhtar MW, Sadaf S,
            Naismith JH
  TITLE     Structural and functional snapshots of a broad-specificity 
            endoglucanase from Thermogutta terrifontis for biomass 
            saccharification.
  JOURNAL   Arch Biochem Biophys. 2025 Feb;764:110274. doi: 
            10.1016/j.abb.2024.110274. Epub 2024 Dec 17.
  PUBMED    39701201
REFERENCE   2  (bases 1 to 6860)
  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: "Arch 
            Biochem Biophys."; date: "2025-02"; volume: "764:110274. doi"; 
            pages: " 10.1016/j.abb.2024.110274. Epub 2024 Dec 17"
COMMENT     Sequence Label: TtEnd_E329A_pEHISTEV
FEATURES             Location/Qualifiers
     source          1..6860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(551..1366)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(551..1366)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1274..1293
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1358..1377
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1488..2076
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1977..1996
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2230..2247
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2262..2401
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2327..2346)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2487..2509
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2503..2694)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3503..4585)
                     /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     4547..4566
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4586..4663)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4869..4888
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4976..4995
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4976..4994
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4995..5019
                     /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)."
     RBS             5034..5056
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5076..5093
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5115..5135
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6696..6713
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6766..6784)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6780..6827
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
      481 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
      541 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
      601 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
      661 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
      721 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
      781 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
      841 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
      901 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
      961 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     1021 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     1081 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     1141 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     1201 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     1261 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     1321 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     1381 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     1441 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     1501 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     1561 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1621 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     1681 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1741 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1801 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1861 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     1921 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     1981 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2041 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     2101 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     2161 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     2221 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     2281 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     2341 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     2401 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2461 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2521 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     2581 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     2641 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     2701 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     2761 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     2821 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     2881 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     2941 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     3001 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     3061 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     3121 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     3181 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     3241 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     3301 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     3361 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     3421 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     3481 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3541 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     3601 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     3661 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     3721 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     3781 agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     3841 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     3901 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     3961 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     4021 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     4081 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     4141 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     4201 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     4261 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     4321 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     4381 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     4441 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     4501 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     4561 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     4621 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     4681 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     4741 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     4801 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     4861 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     4921 ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata
     4981 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     5041 aactttaaga aggagatata catatgtcgt actaccatca ccatcaccat cacgattacg
     5101 atatcccaac gaccgaaaac ctgtattttc agggcgccat ggctgatatc caagaaccgg
     5161 ttaccgttct gcgttgggat tttgaagaaa ttgttggtga acagaatgtt caggcactgc
     5221 ctgcagaatg gattgcaagc agcaaagaag gtattgccgt tgaacgtacc gaacaaggtg
     5281 gtcgttgtct gcatgtttgg gttgatgcag caaccggtcc gggtagccgt aatattcgtt
     5341 atcgtctgcc ggttgataaa ctgcgtggtc agcgtgttcg tgttaatgca ctggttcgtg
     5401 caaaaggtgt tagccagcct ccgaaaccgt ggaatggtat taaatgtatg ctgcgtattg
     5461 aaagcggtgg tgaaattcag tggcctcagc agaatctgcc tggtggtgat tttgattggc
     5521 gtccgattca gtttgttgtt gcagttccgg atgattgtca gcaggttgat ctgattgttg
     5581 gtctggaaaa tgttaccggt gatgcatggt ttgataacat tgcggttgaa gtgatcccga
     5641 aaaaaaagct gagcagcaac cataaagaag tgtttaaagg tcataatctg cctcgtctgc
     5701 gtggtgcaat gattggtccg catgttacca atgccgatct gctggaattt ggtaatgttt
     5761 ggaaagccaa tcatattcgc tggcagctga tttggaatgg ttttccgcat agtccggcag
     5821 atagcgcaac cctggatgaa tatcgtcagt ggctggatgg tgcactgaaa cgtctggaag
     5881 cagcactgcc ggtttgtcgt gaagcaggta ttctggttac cgtggatctg catacaccgc
     5941 ctggtggccg taatgaagca agcgaatgtc gtatttttca cgatcgcgaa tttcagaaag
     6001 cctttattga catctgggaa gatattgcac gtcgttttgc agatagtgat gttgtttggg
     6061 gttatgatct ggttaatgca ccggttgaag gtatggtgcc ggatggtctg atgaattggc
     6121 agcgtctggc cgaagaaacc gcacgtcgtg tgcgtgcaat tgatcagaaa catgcaatta
     6181 tcattgaacc ggcaccgtgg ggtagcccga gcagcattgc tctgctggat ccgattgatg
     6241 ttccgggtgt tgtttatagc gttcatatgt atgttccgca tgcatttacc catcagggtg
     6301 tttatgataa tccggtgggt attgtttatc cgggtacaat tgatggcaaa tggtatgatc
     6361 gtaataccct gcgtaaagtt ctggaaccgg tgcgtcgttt tcaagaagaa aacggggttc
     6421 atatctatat cggtgaattt agcgcaattc gttgggcacc agccgatagc gcatgtcagt
     6481 atctgaaaga ttgcatcgaa atcttcgagg aatatggttg ggattgggca tatcatgcat
     6541 ttcgtgaatg ggatggttgg agcgttgaac atggtccgga tcgtaatgat cgcaatcgta
     6601 ccgcaacacc gaccgatcgt gccctgctgc tgcgtagctg gtatgcagaa aatgtgaaac
     6661 cgcagtttag cgacaaaaag aaggattaac tcgagcacca ccaccaccac cactgagatc
     6721 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     6781 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     6841 ctatatccgg attggcgaat
//
