LOCUS       sequence-318865-        7478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N. tabacum Rubisco small subunit (rbcS-SH1), 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 7478)
  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 7478)
  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-NtXNtS1HR1AtR2NtB2
FEATURES             Location/Qualifiers
     source          1..7478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             379..396
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        477..495
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    496..520
                     /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        1873..1891
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1892..1916
                     /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             1931..1953
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2480..2509
                     /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      2577..2624
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2791..2809
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2810..2834
                     /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             2849..2871
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3125..3154
                     /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      3222..3269
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             3429..3473
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      3525..3572
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3743..4534)
                     /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(4535..4626)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4674..5412)
                     /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(5622..6704)
                     /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     6666..6685
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6705..6782)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        6828..6846
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6847..6871
                     /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        7399..7417
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             7449..7471
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgcaggtgt ggccaccaat taacaagaag aagtacgaga ctctctcata ccttcctgat
       61 ttgagccagg agcaattgct tagtgaagtt gagtaccttt tgaaaaatgg atgggttcct
      121 tgcttggaat tcgagactga gcacggattt gtctatcgtg aaaacaacaa gtcaccagga
      181 tactatgatg gcagatactg gaccatgtgg aagctaccta tgttcggatg cactgatgcc
      241 acccaagtgt tggctgaggt agaagaggcg aagaaggcat acccacaggc ctggatccgt
      301 atcattggat tcgacaacgt gcgtcaagtg cagtgcatca gtttcattgc ctacaagcct
      361 gaaggatatg cgctggaaca ccatcaccat caccattaag gcgcgcccag tgcggccgca
      421 taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat cgaaattaat
      481 acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta
      541 agtataagaa ggagatatac atatgcaaca actttatcag ccttttagac ctcctcctcc
      601 acctctccca cccaaattcc gcaatcttga cacaaatgca aagctcgaag ttctatctaa
      661 tcgccttggt ctttggtatg aatatgctcc tttgatacct tatttaacaa gtgaagggtt
      721 tactccttca actcttgagg aaatcactgg ccttactgga gttgaacaga atcggttggt
      781 tgttgctgcc caagttcgtg atactcttgt tgagtctgct gctttagatg aagaaacgtt
      841 gtcgtatttt gaatctggag gtgctgaatt attatatgaa attcgtttgc ttagtgctag
      901 gcaacgaact gatgcagcaa catttttagt gaagaatggg tttgatgcaa aacaagctca
      961 agatttagct agagcaatta aagattatcc gcgtagacgt gtggattatg gttgggacaa
     1021 gtttaatggt gattctcctg gtgattgttt agcttttatg tactttagat tagctcaaga
     1081 atatgctgcg gcagcttctg aggatttgag gaggtcgtca atggagaagg cattagaagt
     1141 tgttgagtct gaaagtgcga ggaatttgct tgtgatggag ctagaaggac gtgaggttgc
     1201 taaggagagt gtacttgatg atggggtgac agtgcctttg gtgagaatga agttagggga
     1261 ggtagctgag tctacgattg tagtcgtttt gcctgtttgt aaggcggagg gaagggacgt
     1321 ggaggtggag gcggcgccgt gggaatgtgg tggcgtaggg gattttggta tagtggaggc
     1381 ggagaaggat tggaggagat gggttgtttt gcccggatgg caacctatcg cggggttaga
     1441 gagaggtgga gttgcagtgt cgttcaagag cgggaacttt ttgccgtgga gggagaagag
     1501 taagtacaag caagagccgg ttttggtagt ggcggatagg ggaaggacag aagtggcgtc
     1561 ggaggacggg ttttacttgg tggttgatgg tggagacggg agcaatgagg agggattgaa
     1621 agtggaaaga gggtcaacat tgaagaaaag aggggttgaa cagagcttag gaattgtgct
     1681 tatagtagtt aggccaccaa ggtgggaaga tgaagaacaa ttaggtgaag aagattggga
     1741 ttaaagacca agaagcttag ctttctagac tgcaggtcgg cgatataggc gccagcaacc
     1801 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     1861 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     1921 tagaaataat tttgtttaac tttaagaagg agatatacat atgatgtcta atctggcgca
     1981 ggattttctt ggtgacttcg gagctcgtga cccttacccg gaggagatag cgagtcaatt
     2041 cggagataaa gtgttgggat gccaaagcac tgagcacaag attttgatac caaacgcatc
     2101 tgttttgtct ctctcccagc ttcagtgttc ccctgtttcg tcttcacagc ctcctttgtc
     2161 cggcgatgat gccagaactc tcctccacaa ggttttggga tggagtatag tggataatga
     2221 agcgggtggt ctgaaaataa ggtgtatgtg gaaagtgagg gattttgggt gcggtgttga
     2281 actcataaac aggatccata aggttgctga agcttctggt cattaccctt ctcttcactt
     2341 ggaaagtcct acccaagttc gagctgaact atttacctct tctatcggag ggttgagcat
     2401 gaatgatttc ataatggcgg ctaaaataga tgatatcaag acttctgatc tttcccctag
     2461 gaaaagagct tgggcgtgag ctagcatgac tggtggacag caaatgggtc gcggatccgg
     2521 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     2581 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     2641 tatccggata tcccgcaaga ggcccggcag taccggcata accaagcact agtgtcggcg
     2701 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     2761 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
     2821 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat
     2881 gatggccact gacagtgata aagacacgaa agtaaggagc atagtctgtc aaaattgtga
     2941 tggaaatggt gcagtgtcgt gctcacagtg caaaggctct ggggttaact ctgtagatca
     3001 tttcaacggg cggttcaaag ctggtggact atgttggttg tgcagaggta aaaaagatgt
     3061 gttatgtggt gactgcaatg gtgctggatt tcttggtgga tttatgagta cgttcgatga
     3121 gtaggctagc atgactggtg gacagcaaat gggtcgcaga tccggctgct aacaaagccc
     3181 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     3241 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatcccg
     3301 caagaggccc ggcagtaccg gcataaccaa gcggtaccag cggtcaattg tagtggaggt
     3361 gctagcgcat gcggaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg
     3421 agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta
     3481 gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg
     3541 gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca
     3601 cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta
     3661 tttaacgacc ctgccctgaa ccgacgaccg ggtcatcgtg gccggatctt gcggcccctc
     3721 ggcttgaacg aattgttaga cattatttgc cgactacctt ggtgatctcg cctttcacgt
     3781 agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa
     3841 gataagcctg tctagcttca agtatgacgg gctgatactg ggccggcagg cgctccattg
     3901 cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg taccaaatgc
     3961 gggacaacgt aagcactaca tttcgctcat cgccagccca gtcgggcggc gagttccata
     4021 gcgttaaggt ttcatttagc gcctcaaata gatcctgttc aggaaccgga tcaaagagtt
     4081 cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc agcaagatag
     4141 ccagatcaat gtcgatcgtg gctggctcga agatacctgc aagaatgtca ttgcgctgcc
     4201 attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg tcgtcgtgca
     4261 caacaatggt gacttctaca gcgcggagaa tctcgctctc tccaggggaa gccgaagttt
     4321 ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa
     4381 ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag ccgtacaaat
     4441 gtacggccag caacgtcggt tcgagatggc gctcgatgac gccaactacc tctgatagtt
     4501 gagtcgatac ttcggcgatc accgcttccc tcatactctt cctttttcaa tattattgaa
     4561 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4621 aacaaatagc tagctcactc ggtcgctacg ctccgggcgt gagactgcgg cgggcgctgc
     4681 ggacacatac aaagttaccc acagattccg tggataagca ggggactaac atgtgaggca
     4741 aaacagcagg gccgcgccgg tggcgttttt ccataggctc cgccctcctg ccagagttca
     4801 cataaacaga cgcttttccg gtgcatctgt gggagccgtg aggctcaacc atgaatctga
     4861 cagtacgggc gaaacccgac aggacttaaa gatccccacc gtttccggcg ggtcgctccc
     4921 tcttgcgctc tcctgttccg accctgccgt ttaccggata cctgttccgc ctttctccct
     4981 tacgggaagt gtggcgcttt ctcatagctc acacactggt atctcggctc ggtgtaggtc
     5041 gttcgctcca agctgggctg taagcaagaa ctccccgttc agcccgactg ctgcgcctta
     5101 tccggtaact gttcacttga gtccaacccg gaaaagcacg gtaaaacgcc actggcagca
     5161 gccattggta actgggagtt cgcagaggat ttgtttagct aaacacgcgg ttgctcttga
     5221 agtgtgcgcc aaagtccggc tacactggaa ggacagattt ggttgctgtg ctctgcgaaa
     5281 gccagttacc acggttaagc agttccccaa ctgacttaac cttcgatcaa accacctccc
     5341 caggtggttt tttcgtttac agggcaaaag attacgcgca gaaaaaaagg atctcaagaa
     5401 gatcctttga tcttttctac tgaaccgctc tagatttcag tgcaatttat ctcttcaaat
     5461 gtagcacctg aagtcagccc catacgatat aagttgtaat tctcatgtta gtcatgcccc
     5521 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5581 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5641 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5701 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     5761 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     5821 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     5881 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
     5941 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     6001 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     6061 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6121 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6181 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     6241 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6301 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6361 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6421 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6481 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6541 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6601 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6661 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     6721 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     6781 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaaattaa tacgactcac
     6841 tataggggaa ttgtgagcgg ataacaattc ccctgtagaa ataattttgt ttaactttaa
     6901 taaggagata taccatggca aaagcattta agggtaatta caagaattta aagaaggaga
     6961 aaaagagtag gaggaatctt gtggttgtca gtgatttagg tggacagtat gaatatggct
     7021 ttgatgatat caaaacgcaa attcttaact acttcacatt taaagcagtg aggactattt
     7081 tgaaccagct taatgaaatg gactctccac aatatcaatg gttcaatgat tatgttacag
     7141 taaataagct tagggatggg aagcgtttta ttcgcaacct tgctaaggag aagcaagaac
     7201 tcgctgaaaa agtgatggta acacggctca gtctgtatgc gaaatggata aaaaaatgtg
     7261 atcacgagga gatctacaac cgaatatctg atcagaatgt ggaagtgatg cgtgagcggc
     7321 tcatggaaac tgtgatttgg ccatcagatg acacgaactc ggagagagtt gggtgaggcg
     7381 cgcggataac aattgccata atacgactca ctatagggag accacaacgg tttccctcta
     7441 gaaataattt tgtttaactt taagaaggag atatacat
//
