LOCUS       sequence-318862-        7439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N. tabacum Rubisco small subunit (rbcS-Tc), Rubisco 
            chaperone protiens rbcX, raf1, bsd2 and A. thaliana Rubisco 
            chaperone proteins raf2 in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7439)
  AUTHORS   Lin MT, Stone WD, Chaudhari V, Hanson MR
  TITLE     Small subunits can determine enzyme kinetics of tobacco Rubisco 
            expressed in Escherichia coli.
  JOURNAL   Nat Plants. 2020 Oct;6(10):1289-1299. doi: 
            10.1038/s41477-020-00761-5. Epub 2020 Sep 14.
  PUBMED    32929197
REFERENCE   2  (bases 1 to 7439)
  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"; doi: 
            "10.1038/s41477-020-00761-5"; journalName: "Nat Plants"; date: 
            "2020-10"; volume: "6"; issue: "10"; pages: "1289-1299"
COMMENT     Sequence Label: pCDF-NtXNtTcR1AtR2NtB2
FEATURES             Location/Qualifiers
     source          1..7439
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        452..470
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    471..495
                     /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)."
     promoter        1848..1866
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1867..1891
                     /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             1906..1928
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2455..2484
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="ASMTGGQQMG"
     terminator      2552..2599
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2766..2784
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2785..2809
                     /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             2824..2846
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3100..3129
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="ASMTGGQQMG"
     terminator      3197..3244
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             3404..3448
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      3500..3547
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3718..4509)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(4510..4601)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4649..5387)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(5597..6679)
                     /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     6641..6660
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6680..6757)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        6803..6821
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6822..6846
                     /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)."
     promoter        7360..7378
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             7410..7432
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgaagactt ggaatccgat tgacaacaag aaatttgaga cactctcata tctgccacct
       61 ctatcagaag attcgattgc aaaggaggtt gattacatga tccaaaaggg ttggattcct
      121 tgccttgaat ttgatcaggt gggatatgta cgcagggaaa atagcagcat gccaggctac
      181 tatgatggaa ggtattggac actgtggaag ctgcccatgt ttggctgcaa tgactcatcc
      241 caagttctca atgaaatcca agaatgcaag aaagcgtacc caaatgcttt cattcgttgt
      301 ttggcatttg acaatgttaa acaggttcag tgcatggcct ttctcattca gaaacctgca
      361 gcagcatagt acttggcgcg cccagtgcgg ccgcataatg cttaagtcga acagaaagta
      421 atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg ggaattgtga
      481 gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga tatacatatg
      541 caacaacttt atcagccttt tagacctcct cctccacctc tcccacccaa attccgcaat
      601 cttgacacaa atgcaaagct cgaagttcta tctaatcgcc ttggtctttg gtatgaatat
      661 gctcctttga taccttattt aacaagtgaa gggtttactc cttcaactct tgaggaaatc
      721 actggcctta ctggagttga acagaatcgg ttggttgttg ctgcccaagt tcgtgatact
      781 cttgttgagt ctgctgcttt agatgaagaa acgttgtcgt attttgaatc tggaggtgct
      841 gaattattat atgaaattcg tttgcttagt gctaggcaac gaactgatgc agcaacattt
      901 ttagtgaaga atgggtttga tgcaaaacaa gctcaagatt tagctagagc aattaaagat
      961 tatccgcgta gacgtgtgga ttatggttgg gacaagttta atggtgattc tcctggtgat
     1021 tgtttagctt ttatgtactt tagattagct caagaatatg ctgcggcagc ttctgaggat
     1081 ttgaggaggt cgtcaatgga gaaggcatta gaagttgttg agtctgaaag tgcgaggaat
     1141 ttgcttgtga tggagctaga aggacgtgag gttgctaagg agagtgtact tgatgatggg
     1201 gtgacagtgc ctttggtgag aatgaagtta ggggaggtag ctgagtctac gattgtagtc
     1261 gttttgcctg tttgtaaggc ggagggaagg gacgtggagg tggaggcggc gccgtgggaa
     1321 tgtggtggcg taggggattt tggtatagtg gaggcggaga aggattggag gagatgggtt
     1381 gttttgcccg gatggcaacc tatcgcgggg ttagagagag gtggagttgc agtgtcgttc
     1441 aagagcggga actttttgcc gtggagggag aagagtaagt acaagcaaga gccggttttg
     1501 gtagtggcgg ataggggaag gacagaagtg gcgtcggagg acgggtttta cttggtggtt
     1561 gatggtggag acgggagcaa tgaggaggga ttgaaagtgg aaagagggtc aacattgaag
     1621 aaaagagggg ttgaacagag cttaggaatt gtgcttatag tagttaggcc accaaggtgg
     1681 gaagatgaag aacaattagg tgaagaagat tgggattaaa gaccaagaag cttagctttc
     1741 tagactgcag gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     1801 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     1861 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     1921 gaaggagata tacatatgat gtctaatctg gcgcaggatt ttcttggtga cttcggagct
     1981 cgtgaccctt acccggagga gatagcgagt caattcggag ataaagtgtt gggatgccaa
     2041 agcactgagc acaagatttt gataccaaac gcatctgttt tgtctctctc ccagcttcag
     2101 tgttcccctg tttcgtcttc acagcctcct ttgtccggcg atgatgccag aactctcctc
     2161 cacaaggttt tgggatggag tatagtggat aatgaagcgg gtggtctgaa aataaggtgt
     2221 atgtggaaag tgagggattt tgggtgcggt gttgaactca taaacaggat ccataaggtt
     2281 gctgaagctt ctggtcatta cccttctctt cacttggaaa gtcctaccca agttcgagct
     2341 gaactattta cctcttctat cggagggttg agcatgaatg atttcataat ggcggctaaa
     2401 atagatgata tcaagacttc tgatctttcc cctaggaaaa gagcttgggc gtgagctagc
     2461 atgactggtg gacagcaaat gggtcgcgga tccggctgct aacaaagccc gaaaggaagc
     2521 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2581 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc
     2641 ggcagtaccg gcataaccaa gcactagtgt cggcgatata ggcgccagca accgcacctg
     2701 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     2761 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     2821 aattttgttt aactttaaga aggagatata catatgatgg ccactgacag tgataaagac
     2881 acgaaagtaa ggagcatagt ctgtcaaaat tgtgatggaa atggtgcagt gtcgtgctca
     2941 cagtgcaaag gctctggggt taactctgta gatcatttca acgggcggtt caaagctggt
     3001 ggactatgtt ggttgtgcag aggtaaaaaa gatgtgttat gtggtgactg caatggtgct
     3061 ggatttcttg gtggatttat gagtacgttc gatgagtagg ctagcatgac tggtggacag
     3121 caaatgggtc gcagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     3181 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     3241 tttgctgaaa ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata
     3301 accaagcggt accagcggtc aattgtagtg gaggtgctag cgcatgcgga attggatatc
     3361 ggccggccac gcgatcgctg acgtcggtac cctcgagtct ggtaaagaaa ccgctgctgc
     3421 gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa cctaggctgc
     3481 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     3541 ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa
     3601 taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac
     3661 gaccgggtca tcgtggccgg atcttgcggc ccctcggctt gaacgaattg ttagacatta
     3721 tttgccgact accttggtga tctcgccttt cacgtagtgg acaaattctt ccaactgatc
     3781 tgcgcgcgag gccaagcgat cttcttcttg tccaagataa gcctgtctag cttcaagtat
     3841 gacgggctga tactgggccg gcaggcgctc cattgcccag tcggcagcga catccttcgg
     3901 cgcgattttg ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg
     3961 ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc
     4021 aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc gccgctggac ctaccaaggc
     4081 aacgctatgt tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg
     4141 ctcgaagata cctgcaagaa tgtcattgcg ctgccattct ccaaattgca gttcgcgctt
     4201 agctggataa cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg
     4261 gagaatctcg ctctctccag gggaagccga agtttccaaa aggtcgttga tcaaagctcg
     4321 ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg
     4381 cttcaggccg ccatccactg cggagccgta caaatgtacg gccagcaacg tcggttcgag
     4441 atggcgctcg atgacgccaa ctacctctga tagttgagtc gatacttcgg cgatcaccgc
     4501 ttccctcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4561 gagcggatac atatttgaat gtatttagaa aaataaacaa atagctagct cactcggtcg
     4621 ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt tacccacaga
     4681 ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc gccggtggcg
     4741 tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt ttccggtgca
     4801 tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac ccgacaggac
     4861 ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg ttccgaccct
     4921 gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc gctttctcat
     4981 agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg ggctgtaagc
     5041 aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca cttgagtcca
     5101 acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg gagttcgcag
     5161 aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt ccggctacac
     5221 tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt taagcagttc
     5281 cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg tttacagggc
     5341 aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctactgaac
     5401 cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     5461 gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg
     5521 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5581 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5641 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5701 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5761 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5821 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     5881 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5941 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     6001 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     6061 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     6121 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     6181 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     6241 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     6301 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6361 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6421 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     6481 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6541 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     6601 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     6661 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6721 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     6781 tgcgactcct gcattaggaa attaatacga ctcactatag gggaattgtg agcggataac
     6841 aattcccctg tagaaataat tttgtttaac tttaataagg agatatacca tggcaaaagc
     6901 atttaagggt aattacaaga atttaaagaa ggagaaaaag agtaggagga atcttgtggt
     6961 tgtcagtgat ttaggtggac agtatgaata tggctttgat gatatcaaaa cgcaaattct
     7021 taactacttc acatttaaag cagtgaggac tattttgaac cagcttaatg aaatggactc
     7081 tccacaatat caatggttca atgattatgt tacagtaaat aagcttaggg atgggaagcg
     7141 ttttattcgc aaccttgcta aggagaagca agaactcgct gaaaaagtga tggtaacacg
     7201 gctcagtctg tatgcgaaat ggataaaaaa atgtgatcac gaggagatct acaaccgaat
     7261 atctgatcag aatgtggaag tgatgcgtga gcggctcatg gaaactgtga tttggccatc
     7321 agatgacacg aactcggaga gagttgggtg aggcgcgcgt aatacgactc actataggga
     7381 gaccacaacg gtttccctct agaaataatt ttgtttaact ttaagaagga gatatacat
//
