LOCUS       sequence-212058-        4830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin cleavable 
            N-terminal 6His-tag-thioredoxin fusions .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4830)
  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 4830)
  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: pTAT5
FEATURES             Location/Qualifiers
     source          1..4830
                     /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"
     promoter        complement(572..590)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(573..590)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(648..666)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      662..709
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(945..966)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    1085..1106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1119..2201)
                     /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     2163..2182
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2202..2279)
                     /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     2425..2444
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2600..2619)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2719..2741
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2779..2797)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2865..2969
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2970..3038,3039..3830)
                     /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      4001..4589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4490..4509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4743..4760
                     /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 tggatatcga attcgtcgac ctcgagggat ccgggccctc
      541 tagatgcggc cgcatgcata agcttgagta ttctatagtg tcacctaaat cccagcttga
      601 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
      661 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
      721 aactatatcc ggataacctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
      781 cagttgcgca gcctgaatgg cgaatggaaa ttgtaaacgt taatattttg ttaaaattcg
      841 cgttaaatat ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
      901 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
      961 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     1021 atggcccact acagcttgca tgcctgcagg tcggaagcat aaagtgtaaa gcctggggtg
     1081 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1141 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1201 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     1261 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     1321 cgaaaatcct gtttgatggt ggttgacggc gggatataac atgagctgtc ttcggtatcg
     1381 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     1441 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     1501 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     1561 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     1621 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     1681 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     1741 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     1801 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     1861 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     1921 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     1981 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     2041 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     2101 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     2161 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     2221 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcaccattc gatggtgtcg
     2281 ggtaccgagc tcgaatttac gtgaaccatc acccaaatca agttttttgc ggtcgaggtg
     2341 ccgtaaagct ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     2401 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     2461 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     2521 acagggcgcg tcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2581 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     2641 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2701 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2761 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     2821 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2881 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2941 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3001 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3061 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3121 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3181 aagttctgct atgtcataca ctattatccc gtattgacgc cgggcaagag caactcggtc
     3241 gccgggcgcg gtattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3301 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3361 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3421 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3481 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3541 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3601 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3661 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3721 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3781 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3841 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3901 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3961 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4021 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4081 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4141 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4201 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4261 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4321 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4381 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4441 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4501 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4561 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4621 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4681 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4741 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     4801 cgcgttggcc gattcattaa tgcagctgat
//
