LOCUS       sequence-419602-        5823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of Mesembryanthemum crystallinum Thioredoxin F 
            with thrombin-cleavable N-terminal 6xHis tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5823)
  AUTHORS   Stainbrook SC, Aubuchon LN, Chen A, Johnson E, Si A, Walton L, 
            Ahrendt A, Jez J
  TITLE     C4 Grasses Employ Various Strategies to Acclimate Rubisco Activase 
            to Heat Stress
  JOURNAL   bioRxiv 2022.08.24.505184
REFERENCE   2  (bases 1 to 5823)
  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: "bioRxiv 
            2022.08.24.505184"
COMMENT     Sequence Label: pET28a-6xHis-TrxF
FEATURES             Location/Qualifiers
     source          1..5823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..41
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(35..226)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1035..2117)
                     /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     2079..2098
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2118..2195)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2401..2420
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2504..2523
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2504..2522
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2523..2547
                     /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             2562..2584
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2603..2620
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2630..2647
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3191..3208
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3261..3279)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3275..3322
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3359..3814
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3446..3465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3656..3677
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3906..4721)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3906..4721)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4629..4648
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4713..4732
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4843..5431
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5332..5351
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5585..5602
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5617..5756
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5682..5701)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
       61 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
      121 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
      181 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
      241 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
      301 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
      361 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
      421 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
      481 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
      541 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
      601 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
      661 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
      721 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
      781 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
      841 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
      901 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
      961 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     1021 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     1081 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     1141 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     1201 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     1261 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     1321 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     1381 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     1441 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     1501 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     1561 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     1621 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     1681 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     1741 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     1801 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     1861 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     1921 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     1981 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     2041 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     2101 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     2161 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     2221 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     2281 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     2341 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     2401 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     2461 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     2521 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     2581 gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg
     2641 cggcagccat atggctagca gcccgattac tagctttgat ccagcccgta gcccccctaa
     2701 gcgcgttgaa cttggtccta attgccttaa tggtggtgcc acggcaggaa aattgatgcg
     2761 cgagaaggtg ggcgaacgta tgcgtatgtc cgggcgttca tgttgcgtca aggcatcctt
     2821 ggagacagcc gttggggcgg agtccgagac attggttggg aaagtaacag aagtggataa
     2881 ggacacattt tggccgattg ccaacggggc tggggacaag cctgttgtac tggacatgta
     2941 tacacaatgg tgtggtcctt gtaaagtgat ggccccaaaa tatcaagagt tggcagaaaa
     3001 gttgttagac gtagtgttct tgaaattgga ctgtaatcaa gaaaataagc cgttagccaa
     3061 ggaactgggt attcgtgtgg ttcctacgtt caaaatctta aagggaggaa agatcgtaga
     3121 tgaggtcaca ggcgctaagt ttgataaact tgtcgcggcg atcgaagcag ctcgttcatc
     3181 ctgactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     3241 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     3301 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     3361 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3421 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3481 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3541 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3601 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3661 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     3721 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3781 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     3841 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     3901 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     3961 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     4021 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     4081 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     4141 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     4201 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     4261 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     4321 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     4381 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     4441 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     4501 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     4561 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     4621 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     4681 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     4741 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     4801 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4861 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4921 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4981 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5041 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5101 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5161 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5221 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5281 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5341 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5401 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5461 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5521 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5581 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     5641 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     5701 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     5761 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5821 aca
//
