LOCUS       sequence-255698-        6062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and TF (Trigger factor (tig) from E. coli linked to
            Sumo) fusions with optional C-terminal StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6062)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6062)
  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: 
            "unpublished"
COMMENT     Sequence Label: pExp-TFSumo
FEATURES             Location/Qualifiers
     source          1..6062
                     /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..1423
                     /codon_start=1
                     /product="ribosome-associated chaperone from E. coli 
                     (Hoffmann et al., 2010)"
                     /label=Trigger Factor
                     /translation="QVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRK
                     GKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTY
                     SVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRV
                     TIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHA
                     ENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAI
                     RNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQA
                     KRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRN
                     VALEEQAVEAVLAKAKVTEKETTFNELMNQQA"
     CDS             1779..1802
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(1816..1834)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1817..1834)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1892..1910)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1906..1953
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2189..2210)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2360..3442)
                     /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     3404..3423
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3443..3520)
                     /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     3659..3678
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3832..3851)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3951..3973
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4011..4029)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4097..4201
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4202..4270,4271..5062)
                     /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(4420..4439)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5233..5821
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5722..5741
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5975..5992
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc caagtttcag ttgaaaccac tcaaggcctt ggccgccgtg taacgattac
      181 tatcgctgct gacagcatcg aaaccgctgt taaaagcgag ctggtcaacg ttgcgaaaaa
      241 agtacgtatt gacggcttcc gcaaaggcaa agtgccaatg aatatcgttg ctcagcgtta
      301 tggcgcgtct gtacgccagg acgttctggg tgacctgatg agccgtaact tcattgacgc
      361 catcattaaa gaaaaaatca atccggctgg cgcaccgact tatgttccgg gcgaatacaa
      421 gctgggtgaa gacttcactt actctgtaga gtttgaagtt tatccggaag ttgaactgca
      481 gggtctggaa gcgatcgaag ttgaaaaacc gatcgttgaa gtgaccgacg ctgacgttga
      541 cggcatgctg gatactctgc gtaaacagca ggcgacctgg aaagaaaaag acggcgctgt
      601 tgaagcagaa gaccgcgtaa ccatcgactt caccggttct gtagacggcg aagagttcga
      661 aggcggtaaa gcgtctgatt tcgtactggc gatgggccag ggtcgtatga tcccgggctt
      721 tgaagacggt atcaaaggcc acaaagctgg cgaagagttc accatcgacg tgaccttccc
      781 ggaagaatac cacgcagaaa acctgaaagg taaagcagcg aaattcgcta tcaacctgaa
      841 gaaagttgaa gagcgtgaac tgccggaact gactgcagaa ttcatcaaac gtttcggcgt
      901 tgaagatggt tccgtagaag gtctgcgcgc tgaagtgcgt aaaaacatgg agcgcgagct
      961 gaagagcgcc atccgtaacc gcgttaagtc tcaggcgatc gaaggtctgg taaaagctaa
     1021 cgacatcgac gtaccggctg cgctgatcga cagcgaaatc gacgttctgc gtcgccaggc
     1081 tgcacagcgt ttcggtggca acgaaaaaca agctctggaa ctgccgcgcg aactgttcga
     1141 agaacaggct aaacgccgcg tagttgttgg cctgctgctg ggcgaagtta tccgcaccaa
     1201 cgagctgaaa gctgacgaag agcgcgtgaa aggcctgatc gaagagatgg cttctgcgta
     1261 cgaagatccg aaagaagtta tcgagttcta cagcaaaaac aaagaactga tggacaacat
     1321 gcgcaatgtt gctctggaag aacaggctgt tgaagctgta ctggcgaaag cgaaagtgac
     1381 tgaaaaagaa accactttca acgagctgat gaaccagcag gctagcggca gcgatagcga
     1441 agtgaaccag gaagcgaaac cggaagttaa acctgaagtg aagccggaaa cccatattaa
     1501 cctgaaagtg tctgatggca gcagcgaaat cttcttcaaa atcaaaaaaa ccactccgct
     1561 gcgtcgtctg atggaagcgt ttgcgaaacg tcagggcaaa gaaatggata gcctgcgttt
     1621 tctgtatgat ggcattcgta ttcaggcgga tcagactccg gaagatctgg atatggaaga
     1681 taacgatatt atcgaagcgc atcgtgagca gattggtggt acctccggtt ctactgaaaa
     1741 cctgtacttc cagtgagacc ttaattaact cgagcgcatg gagccacccg cagttcgaaa
     1801 aataagcttg agtattctat agtgtcacct aaatcccagc ttgatccggc tgctaacaaa
     1861 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1921 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggataa
     1981 cctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     2041 atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2101 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2161 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2221 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cacagcttgc
     2281 atgcctgcag gtcggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     2341 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2401 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     2461 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     2521 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     2581 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     2641 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     2701 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     2761 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     2821 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     2881 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     2941 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     3001 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     3061 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     3121 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     3181 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     3241 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     3301 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     3361 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     3421 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     3481 atgccatacc gcgaaaggtt ttgcaccatt cgatggtgtc ggcgagctcg aattgtgaac
     3541 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3601 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3661 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3721 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcctga tgcggtattt
     3781 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     3841 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     3901 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3961 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4021 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4081 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4141 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4201 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4261 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4321 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4381 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4441 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4501 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4561 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4621 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4681 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4741 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4801 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4861 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggttc
     4921 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4981 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5041 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5101 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5161 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5221 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5281 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5341 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5401 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5461 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5521 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5581 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5641 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5701 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5761 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5821 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5881 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5941 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6001 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     6061 at
//
