LOCUS       sequence-318851-        7456 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N. tabacum Rubisco small subunit (rbcS-T4a), 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 7456)
  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 7456)
  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-NtXNtT4aR1AtR2NtB2
FEATURES             Location/Qualifiers
     source          1..7456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        455..473
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    474..498
                     /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        1851..1869
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1870..1894
                     /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             1909..1931
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2458..2487
                     /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      2555..2602
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2769..2787
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2788..2812
                     /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             2827..2849
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3103..3132
                     /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      3200..3247
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             3407..3451
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      3503..3550
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3721..4512)
                     /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(4513..4604)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4652..5390)
                     /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(5600..6682)
                     /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     6644..6663
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6683..6760)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        6806..6824
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6825..6849
                     /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        7377..7395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             7427..7449
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgcaggtgt ggccaccaat taacaagaag aagtacgaga cactctcata ccttcctgat
       61 ttgagcgagg agcaattgct tagggaagtt gaataccttt tgaaaaatgg atgggttcct
      121 tgcttggaat tcgagactga gcacggattc gtctaccgtg agaataacaa gtcaccaggt
      181 tactacgatg gaaggtactg gaccatgagg aagttgccca tgttcgggtg cactgatgcc
      241 actcaggtct tggctgaggt cgaggaggca aagaaggctt acccacaagc ctggatcaga
      301 atcattggat tcgacaacgt ccgtcaagtg caatgcatca gtttcatcgc ctacaagccc
      361 gcaggctact aatacttggc gcgcccagtg cggccgcata atgcttaagt cgaacagaaa
      421 gtaatcgtat tgtacacggc cgcataatcg aaattaatac gactcactat aggggaattg
      481 tgagcggata acaattcccc atcttagtat attagttaag tataagaagg agatatacat
      541 atgcaacaac tttatcagcc ttttagacct cctcctccac ctctcccacc caaattccgc
      601 aatcttgaca caaatgcaaa gctcgaagtt ctatctaatc gccttggtct ttggtatgaa
      661 tatgctcctt tgatacctta tttaacaagt gaagggttta ctccttcaac tcttgaggaa
      721 atcactggcc ttactggagt tgaacagaat cggttggttg ttgctgccca agttcgtgat
      781 actcttgttg agtctgctgc tttagatgaa gaaacgttgt cgtattttga atctggaggt
      841 gctgaattat tatatgaaat tcgtttgctt agtgctaggc aacgaactga tgcagcaaca
      901 tttttagtga agaatgggtt tgatgcaaaa caagctcaag atttagctag agcaattaaa
      961 gattatccgc gtagacgtgt ggattatggt tgggacaagt ttaatggtga ttctcctggt
     1021 gattgtttag cttttatgta ctttagatta gctcaagaat atgctgcggc agcttctgag
     1081 gatttgagga ggtcgtcaat ggagaaggca ttagaagttg ttgagtctga aagtgcgagg
     1141 aatttgcttg tgatggagct agaaggacgt gaggttgcta aggagagtgt acttgatgat
     1201 ggggtgacag tgcctttggt gagaatgaag ttaggggagg tagctgagtc tacgattgta
     1261 gtcgttttgc ctgtttgtaa ggcggaggga agggacgtgg aggtggaggc ggcgccgtgg
     1321 gaatgtggtg gcgtagggga ttttggtata gtggaggcgg agaaggattg gaggagatgg
     1381 gttgttttgc ccggatggca acctatcgcg gggttagaga gaggtggagt tgcagtgtcg
     1441 ttcaagagcg ggaacttttt gccgtggagg gagaagagta agtacaagca agagccggtt
     1501 ttggtagtgg cggatagggg aaggacagaa gtggcgtcgg aggacgggtt ttacttggtg
     1561 gttgatggtg gagacgggag caatgaggag ggattgaaag tggaaagagg gtcaacattg
     1621 aagaaaagag gggttgaaca gagcttagga attgtgctta tagtagttag gccaccaagg
     1681 tgggaagatg aagaacaatt aggtgaagaa gattgggatt aaagaccaag aagcttagct
     1741 ttctagactg caggtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     1801 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     1861 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     1921 taagaaggag atatacatat gatgtctaat ctggcgcagg attttcttgg tgacttcgga
     1981 gctcgtgacc cttacccgga ggagatagcg agtcaattcg gagataaagt gttgggatgc
     2041 caaagcactg agcacaagat tttgatacca aacgcatctg ttttgtctct ctcccagctt
     2101 cagtgttccc ctgtttcgtc ttcacagcct cctttgtccg gcgatgatgc cagaactctc
     2161 ctccacaagg ttttgggatg gagtatagtg gataatgaag cgggtggtct gaaaataagg
     2221 tgtatgtgga aagtgaggga ttttgggtgc ggtgttgaac tcataaacag gatccataag
     2281 gttgctgaag cttctggtca ttacccttct cttcacttgg aaagtcctac ccaagttcga
     2341 gctgaactat ttacctcttc tatcggaggg ttgagcatga atgatttcat aatggcggct
     2401 aaaatagatg atatcaagac ttctgatctt tcccctagga aaagagcttg ggcgtgagct
     2461 agcatgactg gtggacagca aatgggtcgc ggatccggct gctaacaaag cccgaaagga
     2521 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     2581 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg
     2641 cccggcagta ccggcataac caagcactag tgtcggcgat ataggcgcca gcaaccgcac
     2701 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     2761 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     2821 aataattttg tttaacttta agaaggagat atacatatga tggccactga cagtgataaa
     2881 gacacgaaag taaggagcat agtctgtcaa aattgtgatg gaaatggtgc agtgtcgtgc
     2941 tcacagtgca aaggctctgg ggttaactct gtagatcatt tcaacgggcg gttcaaagct
     3001 ggtggactat gttggttgtg cagaggtaaa aaagatgtgt tatgtggtga ctgcaatggt
     3061 gctggatttc ttggtggatt tatgagtacg ttcgatgagt aggctagcat gactggtgga
     3121 cagcaaatgg gtcgcagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     3181 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     3241 ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc
     3301 ataaccaagc ggtaccagcg gtcaattgta gtggaggtgc tagcgcatgc ggaattggat
     3361 atcggccggc cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag aaaccgctgc
     3421 tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc
     3481 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     3541 gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt
     3601 caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc
     3661 gacgaccggg tcatcgtggc cggatcttgc ggcccctcgg cttgaacgaa ttgttagaca
     3721 ttatttgccg actaccttgg tgatctcgcc tttcacgtag tggacaaatt cttccaactg
     3781 atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga taagcctgtc tagcttcaag
     3841 tatgacgggc tgatactggg ccggcaggcg ctccattgcc cagtcggcag cgacatcctt
     3901 cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt
     3961 tcgctcatcg ccagcccagt cgggcggcga gttccatagc gttaaggttt catttagcgc
     4021 ctcaaataga tcctgttcag gaaccggatc aaagagttcc tccgccgctg gacctaccaa
     4081 ggcaacgcta tgttctcttg cttttgtcag caagatagcc agatcaatgt cgatcgtggc
     4141 tggctcgaag atacctgcaa gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg
     4201 cttagctgga taacgccacg gaatgatgtc gtcgtgcaca acaatggtga cttctacagc
     4261 gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc
     4321 tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc agcaaatcaa tatcactgtg
     4381 tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt acggccagca acgtcggttc
     4441 gagatggcgc tcgatgacgc caactacctc tgatagttga gtcgatactt cggcgatcac
     4501 cgcttccctc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4561 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagcta gctcactcgg
     4621 tcgctacgct ccgggcgtga gactgcggcg ggcgctgcgg acacatacaa agttacccac
     4681 agattccgtg gataagcagg ggactaacat gtgaggcaaa acagcagggc cgcgccggtg
     4741 gcgtttttcc ataggctccg ccctcctgcc agagttcaca taaacagacg cttttccggt
     4801 gcatctgtgg gagccgtgag gctcaaccat gaatctgaca gtacgggcga aacccgacag
     4861 gacttaaaga tccccaccgt ttccggcggg tcgctccctc ttgcgctctc ctgttccgac
     4921 cctgccgttt accggatacc tgttccgcct ttctccctta cgggaagtgt ggcgctttct
     4981 catagctcac acactggtat ctcggctcgg tgtaggtcgt tcgctccaag ctgggctgta
     5041 agcaagaact ccccgttcag cccgactgct gcgccttatc cggtaactgt tcacttgagt
     5101 ccaacccgga aaagcacggt aaaacgccac tggcagcagc cattggtaac tgggagttcg
     5161 cagaggattt gtttagctaa acacgcggtt gctcttgaag tgtgcgccaa agtccggcta
     5221 cactggaagg acagatttgg ttgctgtgct ctgcgaaagc cagttaccac ggttaagcag
     5281 ttccccaact gacttaacct tcgatcaaac cacctcccca ggtggttttt tcgtttacag
     5341 ggcaaaagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctactg
     5401 aaccgctcta gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca
     5461 tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac
     5521 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5581 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5641 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5701 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5761 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5821 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5881 tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5941 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     6001 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     6061 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     6121 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     6181 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     6241 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     6301 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     6361 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     6421 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     6481 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     6541 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     6601 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6661 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     6721 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6781 ttatgcgact cctgcattag gaaattaata cgactcacta taggggaatt gtgagcggat
     6841 aacaattccc ctgtagaaat aattttgttt aactttaata aggagatata ccatggcaaa
     6901 agcatttaag ggtaattaca agaatttaaa gaaggagaaa aagagtagga ggaatcttgt
     6961 ggttgtcagt gatttaggtg gacagtatga atatggcttt gatgatatca aaacgcaaat
     7021 tcttaactac ttcacattta aagcagtgag gactattttg aaccagctta atgaaatgga
     7081 ctctccacaa tatcaatggt tcaatgatta tgttacagta aataagctta gggatgggaa
     7141 gcgttttatt cgcaaccttg ctaaggagaa gcaagaactc gctgaaaaag tgatggtaac
     7201 acggctcagt ctgtatgcga aatggataaa aaaatgtgat cacgaggaga tctacaaccg
     7261 aatatctgat cagaatgtgg aagtgatgcg tgagcggctc atggaaactg tgatttggcc
     7321 atcagatgac acgaactcgg agagagttgg gtgaggcgcg cggataacaa ttgccataat
     7381 acgactcact atagggagac cacaacggtt tccctctaga aataattttg tttaacttta
     7441 agaaggagat atacat
//
