LOCUS       sequence-212059-        4860 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 4860)
  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 4860)
  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..4860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             98..115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             125..451
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             482..499
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             506..526
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(602..620)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(603..620)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(678..696)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      692..739
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(975..996)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    1115..1136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1149..2231)
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2193..2212
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2232..2309)
                     /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     2455..2474
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2630..2649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2749..2771
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2809..2827)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2895..2999
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3000..3068,3069..3860)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCHTLLSRIDAGQEQLGRRARYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4031..4619
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4520..4539
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4773..4790
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa caccattcat caccatcacc atcacaacac
      121 tagtatgagc gataaaatta ttcacctgac tgacgacagt tttgacacgg atgtactcaa
      181 agcggacggg gcgatcctcg tcgatttctg ggcagagtgg tgcggtccgt gcaaaatgat
      241 cgccccgatt ctggatgaaa tcgctgacga atatcagggc aaactgaccg ttgcaaaact
      301 gaacatcgat caaaaccctg gcactgcgcc gaaatatggc atccgtggta tcccgactct
      361 gctgctgttc aaaaacggtg aagtggcggc aaccaaagtg ggtgcactgt ctaaaggtca
      421 gttgaaagag ttcctcgacg ctaacctggc gactagtgga tctggtggtg gtggcggccg
      481 cctggttccg aggggatcca tgtctgaaaa cctgtacttc cagggttcca tggatatcga
      541 attcgtcgac ctcgagggat ccgggccctc tagatgcggc cgcatgcata agcttgagta
      601 ttctatagtg tcacctaaat cccagcttga tccggctgct aacaaagccc gaaaggaagc
      661 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      721 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggataacctg gcgtaatagc
      781 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa
      841 ttgtaaacgt taatattttg ttaaaattcg cgttaaatat ttgttaaatc agctcatttt
      901 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
      961 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     1021 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acagcttgca tgcctgcagg
     1081 tcggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     1141 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1201 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     1261 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     1321 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttgacggc
     1381 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     1441 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     1501 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     1561 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     1621 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     1681 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     1741 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     1801 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     1861 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     1921 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     1981 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     2041 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     2101 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     2161 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     2221 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     2281 cgaaaggttt tgcaccattc gatggtgtcg ggtaccgagc tcgaatttac gtgaaccatc
     2341 acccaaatca agttttttgc ggtcgaggtg ccgtaaagct ctaaatcgga accctaaagg
     2401 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     2461 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     2521 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcgcctgatg cggtattttc
     2581 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     2641 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     2701 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     2761 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     2821 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     2881 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2941 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3001 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3061 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3121 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3181 aagaacgttt tccaatgatg agcactttta aagttctgct atgtcataca ctattatccc
     3241 gtattgacgc cgggcaagag caactcggtc gccgggcgcg gtattctcag aatgacttgg
     3301 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3361 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3421 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3481 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3541 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3601 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3661 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3721 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3781 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3841 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3901 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3961 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4021 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4081 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4141 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     4201 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4261 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4321 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4381 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4441 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4501 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4561 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4621 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4681 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4741 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4801 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgat
//
