LOCUS       sequence-357154-        6860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TtEnd_E448A_pEHISTEV is a mutant form of TtEnd5A_pEHISTEV vector. It
            expresess mutated TtEnd5A in which Glu448 has been mutated to 
            Ala448. .
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_E448A_pEHISTEV
FEATURES             Location/Qualifiers
     source          1..6860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      30..485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(117..136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     327..348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(577..1392)
                     /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(577..1392)
                     /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     1300..1319
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1384..1403
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1514..2102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2003..2022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2256..2273
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2288..2427
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2353..2372)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2513..2535
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2529..2720)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3529..4611)
                     /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     4573..4592
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4612..4689)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4895..4914
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5002..5021
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5002..5020
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5021..5045
                     /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             5060..5082
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5102..5119
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5141..5161
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6722..6739
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6792..6810)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6806..6853
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
       61 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
      121 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
      181 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
      241 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
      301 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
      361 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
      421 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
      481 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
      541 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
      601 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
      661 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
      721 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
      781 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
      841 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
      901 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
      961 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     1021 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     1081 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     1141 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     1201 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     1261 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     1321 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     1381 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     1441 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1501 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1561 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1621 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     1681 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     1741 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     1801 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     1861 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     1921 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     1981 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2041 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2101 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2161 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     2221 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     2281 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     2341 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     2401 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     2461 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     2521 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     2581 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     2641 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     2701 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     2761 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     2821 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     2881 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     2941 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     3001 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     3061 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     3121 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     3181 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     3241 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     3301 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     3361 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     3421 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     3481 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     3541 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3601 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     3661 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     3721 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     3781 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     3841 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     3901 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     3961 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     4021 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     4081 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     4141 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     4201 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     4261 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     4321 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     4381 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     4441 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     4501 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     4561 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     4621 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     4681 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     4741 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     4801 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     4861 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     4921 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     4981 tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     5041 attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata tgtcgtacta
     5101 ccatcaccat caccatcacg attacgatat cccaacgacc gaaaacctgt attttcaggg
     5161 cgccatggct gatatccaag aaccggttac cgttctgcgt tgggattttg aagaaattgt
     5221 tggtgaacag aatgttcagg cactgcctgc agaatggatt gcaagcagca aagaaggtat
     5281 tgccgttgaa cgtaccgaac aaggtggtcg ttgtctgcat gtttgggttg atgcagcaac
     5341 cggtccgggt agccgtaata ttcgttatcg tctgccggtt gataaactgc gtggtcagcg
     5401 tgttcgtgtt aatgcactgg ttcgtgcaaa aggtgttagc cagcctccga aaccgtggaa
     5461 tggtattaaa tgtatgctgc gtattgaaag cggtggtgaa attcagtggc ctcagcagaa
     5521 tctgcctggt ggtgattttg attggcgtcc gattcagttt gttgttgcag ttccggatga
     5581 ttgtcagcag gttgatctga ttgttggtct ggaaaatgtt accggtgatg catggtttga
     5641 taacattgcg gttgaagtga tcccgaaaaa aaagctgagc agcaaccata aagaagtgtt
     5701 taaaggtcat aatctgcctc gtctgcgtgg tgcaatgatt ggtccgcatg ttaccaatgc
     5761 cgatctgctg gaatttggta atgtttggaa agccaatcat attcgctggc agctgatttg
     5821 gaatggtttt ccgcatagtc cggcagatag cgcaaccctg gatgaatatc gtcagtggct
     5881 ggatggtgca ctgaaacgtc tggaagcagc actgccggtt tgtcgtgaag caggtattct
     5941 ggttaccgtg gatctgcata caccgcctgg tggccgtaat gaagcaagcg aatgtcgtat
     6001 ttttcacgat cgcgaatttc agaaagcctt tattgacatc tgggaagata ttgcacgtcg
     6061 ttttgcagat agtgatgttg tttggggtta tgatctggtt aatgaaccgg ttgaaggtat
     6121 ggtgccggat ggtctgatga attggcagcg tctggccgaa gaaaccgcac gtcgtgtgcg
     6181 tgcaattgat cagaaacatg caattatcat tgaaccggca ccgtggggta gcccgagcag
     6241 cattgctctg ctggatccga ttgatgttcc gggtgttgtt tatagcgttc atatgtatgt
     6301 tccgcatgca tttacccatc agggtgttta tgataatccg gtgggtattg tttatccggg
     6361 tacaattgat ggcaaatggt atgatcgtaa taccctgcgt aaagttctgg aaccggtgcg
     6421 tcgttttcaa gaagaaaacg gggttcatat ctatatcggt gcatttagcg caattcgttg
     6481 ggcaccagcc gatagcgcat gtcagtatct gaaagattgc atcgaaatct tcgaggaata
     6541 tggttgggat tgggcatatc atgcatttcg tgaatgggat ggttggagcg ttgaacatgg
     6601 tccggatcgt aatgatcgca atcgtaccgc aacaccgacc gatcgtgccc tgctgctgcg
     6661 tagctggtat gcagaaaatg tgaaaccgca gtttagcgac aaaaagaagg attaactcga
     6721 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     6781 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     6841 gaggggtttt ttgctgaaag
//
