LOCUS       sequence-220198-        4852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin and TEV 
            cleavable N-terminal 6His-tag-thioredoxin fusions .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4852)
  AUTHORS   Peranen J, Rikkonen M, Hyvonen M, Kaariainen L
  TITLE     T7 vectors with modified T7lac promoter for expression of proteins 
            in Escherichia coli.
  JOURNAL   Anal Biochem. 1996 May 1;236(2):371-3.
  PUBMED    8660525
REFERENCE   2  (bases 1 to 4852)
  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: "Anal 
            Biochem."; date: "1996-05-1"; volume: "236(2)"; pages: "371-3"
COMMENT     Sequence Label: pTAT6
FEATURES             Location/Qualifiers
     source          1..4852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..45
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        26..44
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    44..68
                     /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             81..103
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             123..140
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             150..476
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             507..524
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             531..551
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(627..645)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(628..645)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(703..721)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      717..764
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1000..1021)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1171..2253)
                     /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     2215..2234
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2254..2331)
                     /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."
     primer_bind     2474..2493
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2647..2666)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2766..2788
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2826..2844)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2912..3016
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3017..3085,3086..3877)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3235..3254)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4048..4636
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4537..4556
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4790..4807
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tggccgattc attaatgcag ctgattaata cgactcacta tagggaattg tgagcggata
       61 acaattcctc tagaaataat tttgtttaac tttaagaagg agatatatcc atgaacacca
      121 ttcatcacca tcaccatcac aacactagta tgagcgataa aattattcac ctgactgacg
      181 acagttttga cacggatgta ctcaaagcgg acggggcgat cctcgtcgat ttctgggcag
      241 agtggtgcgg tccgtgcaaa atgatcgccc cgattctgga tgaaatcgct gacgaatatc
      301 agggcaaact gaccgttgca aaactgaaca tcgatcaaaa ccctggcact gcgccgaaat
      361 atggcatccg tggtatcccg actctgctgc tgttcaaaaa cggtgaagtg gcggcaacca
      421 aagtgggtgc actgtctaaa ggtcagttga aagagttcct cgacgctaac ctggcgacta
      481 gtggatctgg tggtggtggc ggccgcctgg ttccgagggg atccatgtct gaaaacctgt
      541 acttccaggg ttccatggat atcgaattcg tcgacctcga gggatccggg ccctctagat
      601 gcggccgcat gcataagctt gagtattcta tagtgtcacc taaatcccag cttgatccgg
      661 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
      721 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
      781 tatccggata acctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
      841 gcgcagcctg aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta
      901 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
      961 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1021 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1081 ccacagcttg catgcctgca ggtcggaagc ataaagtgta aagcctgggg tgcctaatga
     1141 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1201 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1261 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     1321 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     1381 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     1441 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     1501 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     1561 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     1621 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     1681 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     1741 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     1801 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     1861 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     1921 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     1981 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     2041 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     2101 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     2161 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     2221 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     2281 cgggcgctat catgccatac cgcgaaaggt tttgcaccat tcgatggtgt cgggtaccga
     2341 gctcgaattg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     2401 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     2461 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     2521 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     2581 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     2641 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     2701 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     2761 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     2821 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     2881 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2941 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3001 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3061 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3121 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3181 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3241 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3301 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3361 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3421 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3481 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3541 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     3601 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3661 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3721 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3781 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3841 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3901 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     3961 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4021 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4081 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4141 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4201 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4261 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4321 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4381 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4441 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4501 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4561 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4621 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4681 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4741 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4801 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gt
//
