LOCUS       sequence-357162-        6860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TtEnd5A_pEHISTEV expression vector encodes first Thermogutta 
            terrifontis Endoglucanase (19-531 amino acid residues) of Glycoside 
            Hydrolase Family 5. .
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: TtEnd5A_pEHISTEV
FEATURES             Location/Qualifiers
     source          1..6860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(38..56)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      52..99
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      136..591
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(223..242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     433..454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(683..1498)
                     /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(683..1498)
                     /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     1406..1425
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1490..1509
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1620..2208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2109..2128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2362..2379
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2394..2533
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2459..2478)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2619..2641
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2635..2826)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3635..4717)
                     /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     4679..4698
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4718..4795)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5001..5020
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5108..5127
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5108..5126
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5127..5151
                     /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             5166..5188
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5208..5225
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5247..5267
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6828..6845
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
       61 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
      121 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      181 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      241 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      301 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      361 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      421 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
      481 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
      541 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
      601 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
      661 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
      721 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
      781 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
      841 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
      901 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
      961 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     1021 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     1081 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     1141 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     1201 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     1261 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     1321 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     1381 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     1441 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     1501 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     1561 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1621 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1681 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1741 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1801 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1861 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1921 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1981 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2041 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2101 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2161 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2221 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2281 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2341 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     2401 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     2461 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     2521 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2581 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2641 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2701 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2761 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2821 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2881 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2941 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     3001 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     3061 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3121 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3181 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3241 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3301 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3361 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     3421 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     3481 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     3541 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3601 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3661 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3721 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3781 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3841 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3901 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3961 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     4021 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     4081 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4141 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4201 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4261 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4321 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4381 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4441 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4501 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4561 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4621 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4681 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4741 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4801 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4861 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4921 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4981 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     5041 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc
     5101 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     5161 ataattttgt ttaactttaa gaaggagata tacatatgtc gtactaccat caccatcacc
     5221 atcacgatta cgatatccca acgaccgaaa acctgtattt tcagggcgcc atggctgata
     5281 tccaagaacc ggttaccgtt ctgcgttggg attttgaaga aattgttggt gaacagaatg
     5341 ttcaggcact gcctgcagaa tggattgcaa gcagcaaaga aggtattgcc gttgaacgta
     5401 ccgaacaagg tggtcgttgt ctgcatgttt gggttgatgc agcaaccggt ccgggtagcc
     5461 gtaatattcg ttatcgtctg ccggttgata aactgcgtgg tcagcgtgtt cgtgttaatg
     5521 cactggttcg tgcaaaaggt gttagccagc ctccgaaacc gtggaatggt attaaatgta
     5581 tgctgcgtat tgaaagcggt ggtgaaattc agtggcctca gcagaatctg cctggtggtg
     5641 attttgattg gcgtccgatt cagtttgttg ttgcagttcc ggatgattgt cagcaggttg
     5701 atctgattgt tggtctggaa aatgttaccg gtgatgcatg gtttgataac attgcggttg
     5761 aagtgatccc gaaaaaaaag ctgagcagca accataaaga agtgtttaaa ggtcataatc
     5821 tgcctcgtct gcgtggtgca atgattggtc cgcatgttac caatgccgat ctgctggaat
     5881 ttggtaatgt ttggaaagcc aatcatattc gctggcagct gatttggaat ggttttccgc
     5941 atagtccggc agatagcgca accctggatg aatatcgtca gtggctggat ggtgcactga
     6001 aacgtctgga agcagcactg ccggtttgtc gtgaagcagg tattctggtt accgtggatc
     6061 tgcatacacc gcctggtggc cgtaatgaag caagcgaatg tcgtattttt cacgatcgcg
     6121 aatttcagaa agcctttatt gacatctggg aagatattgc acgtcgtttt gcagatagtg
     6181 atgttgtttg gggttatgat ctggttaatg aaccggttga aggtatggtg ccggatggtc
     6241 tgatgaattg gcagcgtctg gccgaagaaa ccgcacgtcg tgtgcgtgca attgatcaga
     6301 aacatgcaat tatcattgaa ccggcaccgt ggggtagccc gagcagcatt gctctgctgg
     6361 atccgattga tgttccgggt gttgtttata gcgttcatat gtatgttccg catgcattta
     6421 cccatcaggg tgtttatgat aatccggtgg gtattgttta tccgggtaca attgatggca
     6481 aatggtatga tcgtaatacc ctgcgtaaag ttctggaacc ggtgcgtcgt tttcaagaag
     6541 aaaacggggt tcatatctat atcggtgaat ttagcgcaat tcgttgggca ccagccgata
     6601 gcgcatgtca gtatctgaaa gattgcatcg aaatcttcga ggaatatggt tgggattggg
     6661 catatcatgc atttcgtgaa tgggatggtt ggagcgttga acatggtccg gatcgtaatg
     6721 atcgcaatcg taccgcaaca ccgaccgatc gtgccctgct gctgcgtagc tggtatgcag
     6781 aaaatgtgaa accgcagttt agcgacaaaa agaaggatta actcgagcac caccaccacc
     6841 accactgaga tccggctgct
//
