LOCUS       sequence-212041-        4790 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and Trx (thioredoxin (trxA) from E. coli fusions 
            with optional C-terminal StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4790)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4790)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "unpublished"
COMMENT     Sequence Label: pExp-Trx
FEATURES             Location/Qualifiers
     source          1..4790
                     /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             92..115
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             131..454
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEI
                     ADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLD
                     ANLA"
     CDS             507..530
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(544..562)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(545..562)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(620..638)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      634..681
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(917..938)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1088..2170)
                     /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     2132..2151
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2171..2248)
                     /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     2387..2406
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2560..2579)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2679..2701
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2739..2757)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2825..2929
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2930..2998,2999..3790)
                     /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(3148..3167)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3961..4549
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4450..4469
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4703..4720
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc agcgataaaa ttattcacct gactgacgac agttttgaca cggatgtact
      181 caaagcggac ggggcgatcc tcgtcgattt ctgggcagag tggtgcggtc cgtgcaaaat
      241 gatcgccccg attctggatg aaatcgctga cgaatatcag ggcaaactga ccgttgcaaa
      301 actgaacatc gatcaaaacc ctggcactgc gccgaaatat ggcatccgtg gtatcccgac
      361 tctgctgctg ttcaaaaacg gtgaagtggc ggcaaccaaa gtgggtgcac tgtctaaagg
      421 tcagttgaaa gagttcctcg acgctaacct ggccagcggt accgaaaacc tgtacttcca
      481 gtgagacctt aattaactcg agcgcatgga gccacccgca gttcgaaaaa taagcttgag
      541 tattctatag tgtcacctaa atcccagctt gatccggctg ctaacaaagc ccgaaaggaa
      601 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      661 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggataacc tggcgtaata
      721 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
      781 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
      841 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
      901 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
      961 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca cagcttgcat gcctgcaggt
     1021 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactta cattaattgc
     1081 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1141 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     1201 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     1261 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     1321 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     1381 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     1441 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     1501 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     1561 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     1621 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     1681 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     1741 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     1801 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     1861 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     1921 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     1981 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     2041 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     2101 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     2161 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     2221 gaaaggtttt gcaccattcg atggtgtcgg cgagctcgaa ttgtgaacca tcaccctaat
     2281 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2341 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2401 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2461 ccgccgcgct taatgcgccg ctacagggcg cgtcctgatg cggtattttc tccttacgca
     2521 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     2581 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2641 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2701 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     2761 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     2821 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2881 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2941 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3001 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3061 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3121 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     3181 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3241 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3301 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3361 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3421 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3481 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3541 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3601 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttctc gcggtatcat
     3661 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3721 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3781 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3841 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3901 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3961 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4021 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4081 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4141 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4201 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4261 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4321 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4381 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4441 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4501 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4561 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4621 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4681 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     4741 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgat
//
