LOCUS       sequence-360163-        6860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TtEnd_H280A_pEHISTEV is a mutant form of TtEnd5A_pEHISTEV vector. It
            expresess mutated TtEnd5A in which His280 has been mutated with 
            Ala280. .
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_H280A_pEHISTEV
FEATURES             Location/Qualifiers
     source          1..6860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      54..509
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(141..160)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     351..372
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(601..1416)
                     /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(601..1416)
                     /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     1324..1343
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1408..1427
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1538..2126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2027..2046
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2280..2297
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2312..2451
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2377..2396)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2537..2559
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2553..2744)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3553..4635)
                     /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     4597..4616
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4636..4713)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4919..4938
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5026..5045
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5026..5044
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5045..5069
                     /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             5084..5106
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5126..5143
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5165..5185
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6746..6763
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6816..6834)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(6830..6860,1..17)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
       61 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      121 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      181 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      241 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
      301 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg tkctttaata gtggactctt
      361 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
      421 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
      481 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
      541 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
      601 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
      661 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
      721 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
      781 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
      841 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
      901 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
      961 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     1021 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     1081 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     1141 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     1201 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     1261 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     1321 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     1381 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     1441 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     1501 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1561 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1621 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1681 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1741 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1801 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1861 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1921 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1981 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2041 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2101 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2161 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2221 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2281 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2341 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     2401 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     2461 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     2521 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     2581 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     2641 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     2701 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     2761 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     2821 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     2881 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     2941 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3001 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3061 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3121 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     3181 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     3241 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     3301 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     3361 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     3421 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     3481 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     3541 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3601 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     3661 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     3721 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     3781 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     3841 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     3901 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     3961 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     4021 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     4081 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     4141 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     4201 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     4261 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     4321 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     4381 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     4441 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     4501 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     4561 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     4621 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     4681 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     4741 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     4801 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     4861 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     4921 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     4981 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta
     5041 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
     5101 aggagatata catatgtcgt actaccatca ccatcaccat cacgattacg atatcccaac
     5161 gaccgaaaac ctgtattttc agggcgccat ggctgatatc caagaaccgg ttaccgttct
     5221 gcgttgggat tttgaagaaa ttgttggtga acagaatgtt caggcactgc ctgcagaatg
     5281 gattgcaagc agcaaagaag gtattgccgt tgaacgtacc gaacaaggtg gtcgttgtct
     5341 gcatgtttgg gttgatgcag caaccggtcc gggtagccgt aatattcgtt atcgtctgcc
     5401 ggttgataaa ctgcgtggtc agcgtgttcg tgttaatgca ctggttcgtg caaaaggtgt
     5461 tagccagcct ccgaaaccgt ggaatggtat taaatgtatg ctgcgtattg aaagcggtgg
     5521 tgaaattcag tggcctcagc agaatctgcc tggtggtgat tttgattggc gtccgattca
     5581 gtttgttgtt gcagttccgg atgattgtca gcaggttgat ctgattgttg gtctggaaaa
     5641 tgttaccggt gatgcatggt ttgataacat tgcggttgaa gtgatcccga aaaaaaagct
     5701 gagcagcaac cataaagaag tgtttaaagg tcataatctg cctcgtctgc gtggtgcaat
     5761 gattggtccg catgttacca atgccgatct gctggaattt ggtaatgttt ggaaagccaa
     5821 tcatattcgc tggcagctga tttggaatgg ttttccgcat agtccggcag atagcgcaac
     5881 cctggatgaa tatcgtcagt ggctggatgg tgcactgaaa cgtctggaag cagcactgcc
     5941 ggtttgtcgt gaagcaggta ttctggttac cgtggatctg gctacaccgc ctggtggccg
     6001 taatgaagca agcgaatgtc gtatttttca cgatcgcgaa tttcagaaag cctttattga
     6061 catctgggaa gatattgcac gtcgttttgc agatagtgat gttgtttggg gttatgatct
     6121 ggttaatgaa ccggttgaag gtatggtgcc ggatggtctg atgaattggc agcgtctggc
     6181 cgaagaaacc gcacgtcgtg tgcgtgcaat tgatcagaaa catgcaatta tcattgaacc
     6241 ggcaccgtgg ggtagcccga gcagcattgc tctgctggat ccgattgatg ttccgggtgt
     6301 tgtttatagc gttcatatgt atgttccgca tgcatttacc catcagggtg tttatgataa
     6361 tccggtgggt attgtttatc cgggtacaat tgatggcaaa tggtatgatc gtaataccct
     6421 gcgtaaagtt ctggaaccgg tgcgtcgttt tcaagaagaa aacggggttc atatctatat
     6481 cggtgaattt agcgcaattc gttgggcacc agccgatagc gcatgtcagt atctgaaaga
     6541 ttgcatcgaa atcttcgagg aatatggttg ggattgggca tatcatgcat ttcgtgaatg
     6601 ggatggttgg agcgttgaac atggtccgga tcgtaatgat cgcaatcgta ccgcaacacc
     6661 gaccgatcgt gccctgctgc tgcgtagctg gtatgcagaa aatgtgaaac cgcagtttag
     6721 cgacaaaaag aaggattaac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     6781 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     6841 ccttggggcc tctaaacggg
//
