LOCUS       sequence-492819-        9915 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sequential knock out of A. thaliana Rubisco assembly factors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9915)
  AUTHORS   Li S, Lee B, Lao Y, Lertwiriyapiti S, Huang X, Wang T
  TITLE     Directed evolution of a plant Rubisco chaperone with altered client 
            recognition.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Sep 16;122(37):e2510701122. doi: 
            10.1073/pnas.2510701122. Epub 2025 Sep 11.
  PUBMED    40932770
REFERENCE   2  (bases 1 to 9915)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2025-09-16"; volume: "122(37):e2510701122. 
            doi"; pages: " 10.1073/pnas.2510701122. Epub 2025 Sep 11"
COMMENT     Sequence Label: pTW334e3
FEATURES             Location/Qualifiers
     source          1..9915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             86..877
                     /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        join(1194..1199,1200..1215,1216..1222)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1230..1246
                     /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             4050..4072
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             5339..5347
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     RBS             5765..5787
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    7509..7525
                     /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        complement(join(7533..7539,7540..7555,7556..7561))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     promoter        7804..7881
                     /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."
     CDS             7882..8964
                     /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     complement(7901..7920)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      9121..9859
                     /direction=RIGHT
                     /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."
     promoter        join(9909..9915,1..85)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
       61 tcaataatat tgaaaaagga agagtatgag ggaagcggtg atcgccgaag tatcgactca
      121 actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca
      181 tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt
      241 tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga
      301 aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt
      361 gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg
      421 gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc
      481 tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga
      541 actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct
      601 atggaactcg ccgcccgact gggctggaga tgagcgaaat gtagtgctta cgttgtcccg
      661 catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc
      721 aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct
      781 tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta
      841 cgtgaaaggc gagatcacca aggtagtcgg caaataaacg ccatgggcat taaaaaatgc
      901 cctcttgggt tatcaagagg gtcattatat ttgctgagga gtgccgttaa tgcgcaacgc
      961 aattaatgta agttagctca ctcattaggc acaattctca tgtttgacag cttatcatcg
     1021 actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg
     1081 tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa
     1141 tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa
     1201 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     1261 cagccagtcc gtttaggtgt tttcacgagc acttcaccaa caacgcgtac aactaggaga
     1321 tataccatgg gtatgcaaca gctctaccaa ccgttccgac caccgtcatc tcccattcca
     1381 acccaattcc gttcactcga ctccgccggt aaaatcgaaa tcctcgccgg tcgaatggct
     1441 ctctggttcg aatacgcacc tcttatctct tctctttaca ccgatggttt cactcctcca
     1501 accatcgaag aactcaccgg aatctcaagc atcgaacaga accgtttaat cgtcggtgcg
     1561 caagttcgtg actcaattct tcaatctatc catgaaccgg aacttatttc tgcgttcgac
     1621 accggtggtg cagagcttct atacgaaatt cgtctcctta gcaccacgca gcgtgttgca
     1681 gccgccacat tcatcattga tcgtaatatt gattcgaaag gagcacagga tttggcacga
     1741 gccatcaagg attatcctaa ccgtcgtgga gacgttggat ggttggattt tgattacaac
     1801 ttaccaggag attgtctttc attcttgtat tacaggcaga gcagagagaa taagaatcca
     1861 tcggatcaga gaacctcgat gcttctacaa gcattggggg ttgcagaatc agaaaaggct
     1921 aagaacaggt tgaacacaga gctatacggg gacaaggaag cagagaagga gaaggagaag
     1981 aagaagaagg aagaggaagt caaagctatt cggattccag tggtgaggct taagtttgga
     2041 gaagtagcag aagcgacctc ggtggtagtt ttaccggttt gtaaagcaga ggaaggggaa
     2101 aagaagattc ttgaagctcc aatggagatc atagccggag gggattttaa ggtggttgag
     2161 gcggagaaag gatggaagag atgggtagtg cttccgtcgt ggaacccagt ggcagccatt
     2221 gggaaaggcg gtgtggcggt ttctttcagg gatgatagaa aagtgctgcc ttgggatgga
     2281 aaggaggagc ctttactggt agtggccgat agggtgagga atgttgtgga ggctgatgac
     2341 gggtattatc tcgtggtggc tgagaacgga cttaagctag agaaaggatc agatttgaag
     2401 gcgagagagg tgaaggagag tttagggatg gttgttttgg tggtgaggcc gccaagagaa
     2461 gatgatgatg attggcagac aagtcatcag aactgggact gaattacaaa aattaggaga
     2521 tatacatatg atgtctaatc tggcgcagga ttttcttggt gacttcggag ctcgtgaccc
     2581 ttacccggag gagatagcga gtcaattcgg agataaagtg ttgggatgcc aaagcactga
     2641 gcacaagatt ttgataccaa acgcatctgt tttgtctctc tcccagcttc agtgttcccc
     2701 tgtttcgtct tcacagcctc ctttgtccgg cgatgatgcc agaactctcc tccacaaggt
     2761 tttgggatgg agtatagtgg ataatgaagc gggtggtctg aaaataaggt gtatgtggaa
     2821 agtgagggat tttgggtgcg gtgttgaact cataaacagg atccataagg ttgctgaagc
     2881 ttctggtcat tacccttctc ttcacttgga aagtcctacc caagttcgag ctgaactatt
     2941 tacctcttct atcggagggt tgagcatgaa tgatttcata atggcggcta aaatagatga
     3001 tatcaagact tctgatcttt cccctaggaa aagagcttgg gcgtgattca gctaagacac
     3061 tgcacaggag atatacatat gatggccgca aacaataatc ctcagggcac taaaccaaac
     3121 agtttagttt gtgcaaattg tgaaggggaa ggttgtgtgg catgttctca atgcaaagga
     3181 ggtggtgtga atttgattga tcatttcaat ggtcagttta aagctggtgc actctgttgg
     3241 ctttgcagag gtaaaaagga ggtgttgtgt ggggactgca atggagctgg tttcattggt
     3301 gggttcttga gcaccttcga tgagtagagt cagtagggcc ctaaaaaaaa accccgcccc
     3361 tgacagggca gggttttttt tggcgaaaaa accccgctga agcggggttt tttgcgtaca
     3421 ttggctccgt ctaagaaagt atagccatca catctgaagc tctgagggca atgtagttgg
     3481 aaccatcttt gcccttgaag tcgttaccag catacttgga gtaaagtact gtgcttccgg
     3541 ttgatactgg tagaggcgta attttacctt cctcgtctag ggaacccggt ccaactgcta
     3601 tcacggtgcc aatagaaggc ttctctttgg tagtctcggt taacaacaac cctccagctg
     3661 ttttctcctc cgcctcagca accttaataa agactcggtc attcaaaggt ttgagatctt
     3721 tgatgtcctc tgtctcaaga atgccaacaa tatcatcttc cttgaggata agatgcttca
     3781 catcattgaa ctccacctca gttcctgcgt atttggagta gataatttgt gctccagtag
     3841 ggacagtgat atcaattttg ttcttcccaa tagttcttcc ttcacccaca gcaacgactt
     3901 cacctccttg aggttttgat tgagcagtgg atggaagtaa gataccacct aaagtcttct
     3961 cctctgcctc cttgatcttc accaaaactc gatctcccaa tggcttaatt gaagtatact
     4021 taggggcaac aacagaggcc atatgtatat ctccttctta aagttaaaca aaattatttc
     4081 tagagggcaa ttgttatccg gtcgacgtcg accttagtat ccgtaacctg agttgtccat
     4141 tgggttgccg acgggaactg gttcaggttc cttgatctca acaacaacgc agtccgacat
     4201 caagaaagtc tttgcaaccg aagctgcgtg ttccaagcaa catctcacaa cctttgttgg
     4261 atcgatgatt cctgcagcca tcaaatcctc gtacttgccg gttgcagcat tgtaaccgaa
     4321 tttcacgtta tcgttggaaa gcaccttctc gctaactaca cttccattaa caccagcatt
     4381 cttggcgatt agttttaggg ggtaacttag tgctctttta actatatccg ctccaacctt
     4441 ttcttcatca ttatcaagcg tggctttaat ggcatcaacc ttggaagcaa gacgaagcag
     4501 agtgcaacca ccaccaacaa caataccttc ctcgacagcg gcctttgttg cattaagagc
     4561 atcttcaact ctcaacttct tctctttgag ttctgtctca gtttgtgctc caacctgaat
     4621 cacagcaact ccaccagaga gctttgcaat tctctcattg agtttctcct tctcgtaatc
     4681 ttgctccgct tgctcaataa gatttttaat ctgtgtaaca cgctttttca ctgcatcctg
     4741 tgtgcttcca tcacccacta tggtcgaggt ttcctttgtg aggacaacct ttgaagcatt
     4801 accaagaacc tcttttccag ctttgtcaag tgaaagacca acttcctcac ggatcacagt
     4861 tgctccagtt agaatggcaa tatcatcaag gtattggctc ttgcgctctc caaaccctgg
     4921 agctctgaga gctgctatct tcagtgtacc tctaagcttg tttacaacaa gagtcgctaa
     4981 agcttcttgt tcaatgtctt cagcaataat taatattggg tatccgcctc taattgcatc
     5041 ctccaaaaca ccaacaagat cccttgcatt ggtaattttc ttgtcaacga gaagtaactt
     5101 gcaattgtcg aactcgacgg acattttctc gctgtctgtc acaaagtaag gagagatata
     5161 accacggtca aattgcattc cttctacgac gtagaggttg ttctcggcac ttttaccctc
     5221 ctcaagggta accacacctt tcctgcccac tttgctcatt gcttcagcaa tcatatttcc
     5281 aatttcgtcg ttgttacccg cactaacagc tgcaacatct gcaagttcac tgtcttcaac
     5341 ctccttagac attttcttga gctcggtaac caaagccttt gctgtcttct caatacctct
     5401 ggtgatcaaa acagggtttg caccagcagc taccaccttg acaccctcag cgataaaacc
     5461 ttgtgcaaga acaacagatg ttgttgtacc gtcaccagcc aagtcattgg ttttggcagc
     5521 cgcttgcctc acaagcttag cgccaatgtt ctcaaccggg tcttccaact caacctctct
     5581 tgcaacagtc acaccatcat taacaattcg tggtgatcca tacttgctct caagaacaac
     5641 atttcgtcct ttgggtccaa gtgtaacgcc aacaagatct gcgagcttgt tgacaccagc
     5701 ttgaagtctc ctaatggtag tcccatcttt gttgaaatgt aattcctttg ctgccatatg
     5761 tatatctcct tcttaaagtt aaacaaaatt atttctagct ccttacacca tgaggccctc
     5821 aggagcagca gcagcaggag ccttgggttt cggtttgtca acaacaatgg cctgagtggt
     5881 cagtaccatt cctgctacgg aagcagcgtt ctgtagcgca catcttgtca ctttagccgg
     5941 atcaatcact ccagcttcga acagattctc gtaagtatca gtcattgcgt tgtacccatt
     6001 ctcccaatcg ctgaacataa tcttttccac aacaacttct ccttcaactc cagcgttctg
     6061 agcaataaga gcagctggtg acagcaatgc cttttgtact atgtcagctc ccaaacgttc
     6121 atcagcatcc tcaaaagtct ccttaatggc gggaataaca gtggagagat gcaccaaagc
     6181 agcaccacca cctggaacta tgccttcctc gatagcagca aatgttgcgt tctttgcatc
     6241 ctcgatacga agcttacggt cctcaagctc agtttcagtt gctgctccga ctttaatgac
     6301 agcaacacct ccagatagct tcgcaattct ctcagcgagc ttctctgagt catacacaga
     6361 atcagtctca aatagttctt tcttcagctg agcaatccga gcttgtaatt cgtccttgga
     6421 agctgcatct gcaatgaggg tagtcgaatc tttgctaatc gtgacttttc gggcaatacc
     6481 caattgatct atggttgcgt tttcaaccag taagctcatg tccatggcta ggtactcagc
     6541 tcctgtcaag attgcaatat cttgaagcat ggcttttctt ctttctccaa atcctggcgc
     6601 tttaacggca acaacattga ggacaccacg aagcttgttc acgacaaggg tagctaaggc
     6661 ttcaccagta acatcctctg caataatcaa caacggagca cgaagctgag tggtcttctc
     6721 caaaataggg atgatgtctt tgatcgcagt gatcttctga tcagtgatca ataccctagc
     6781 attctcaaac tcagctagta gtttctcagg gtttgtaaca aactgaggcg agatgtaacc
     6841 tctgtcaatc tccatccctt cttcaacttc gaccgtggtt tcaaaagaag atgaagattc
     6901 aatggacaaa acaccatcag gtccaacttt gtcgatggca tcagcaatca ttgaccctat
     6961 aaggtcatca tttccagcag agatagaagc cacagctctg atgtcatcac gacctttcac
     7021 gggcctagct ttcttctgaa gctcttcgat cagaccttga acagtcttat caattcccct
     7081 cttgagtgaa acgggattcg caccagaagt gacgctcaac aatccatgtt tgattatctc
     7141 ccgagcaagg atagacgcag tggttgtccc atcaccagca gagtcattag tcttactcgc
     7201 aacctcacga ataagcgctg caccagcatt ttccatggca ttaggtaact caatagctct
     7261 agcaatggtg actccatcat tcacaacctt aggacttcca aattcatcca acacaacatt
     7321 cctccctctg gggccaagcg taagaccaac acaatcagca agcttatcaa taccagcttg
     7381 tagagcagct ctagagtgct ggtcaaaagc tatttccttt acattcatat gtatatctcc
     7441 tactgcacct attgttggtg aagtgctcgt gaaaacacct aaacggactg gctgtttcct
     7501 gtgtgaaatt gttatccgct cacaattcca cacattatac gagccgatga ttaattgtca
     7561 acagctcatt tcagaatatt tgccagaacc gttatgatgt cggcgcaaaa aacattatcc
     7621 agaacgggag tgcgccttga gcgacacgaa ttatgcagtg atttacgacc tgcacagcca
     7681 taccacagct tccgatggct gcctgacgcc agaagcattg gtgcaccgtg cagtcgatga
     7741 taagctgtca aacatgagaa ttgtgcctaa tgagtgagct aacttacatt aattgcgttg
     7801 cgcgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     7861 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     7921 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     7981 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     8041 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     8101 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     8161 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     8221 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     8281 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     8341 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     8401 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     8461 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     8521 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     8581 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     8641 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     8701 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     8761 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     8821 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     8881 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     8941 tcccgactgg aaagcgggca gtgaagggtc cctaagtctc ctcagcaaaa cgaaaggccc
     9001 agtctttcga ctgagccttt cgttttattt gaccggatgt cctcttcctc ctgggctgac
     9061 ttcaggtgct acatttgaag agataaattg cactgaaatc tagagcggtt cagtagaaaa
     9121 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atcttttgcc ctgtaaacga
     9181 aaaaaccacc tggggaggtg gtttgatcga aggttaagtc agttggggaa ctgcttaacc
     9241 gtggtaactg gctttcgcag agcacagcaa ccaaatctgt ccttccagtg tagccggact
     9301 ttggcgcaca cttcaagagc aaccgcgtgt ttagctaaac aaatcctctg cgaactccca
     9361 gttaccaatg gctgctgcca gtggcgtttt accgtgcttt tccgggttgg actcaagtga
     9421 acagttaccg gataaggcgc agcagtcggg ctgaacgggg agttcttgct tacagcccag
     9481 cttggagcga acgacctaca ccgagccgag ataccagtgt gtgagctatg agaaagcgcc
     9541 acacttcccg taagggagaa aggcggaaca ggtatccggt aaacggcagg gtcggaacag
     9601 gagagcgcaa gagggagcga cccgccggaa acggtgggga tctttaagtc ctgtcgggtt
     9661 tcgcccgtac tgtcagattc atggttgagc ctcacggctc ccacagatgc accggaaaag
     9721 cgtctgttta tgtgaactct ggcaggaggg cggagcctat ggaaaaacgc caccggcgcg
     9781 gccctgctgt tttgcctcac atgttagtcc cctgcttatc cacggaatct gtgggtaact
     9841 ttgtatgtgt ccgcagcgcc cgccgcagtc tcacgcccgg agcgtagcga ccgagtgacg
     9901 gccttaattt ggctt
//
