LOCUS       sequence-490609-        9828 bp    DNA     circular SYN 24-OCT-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 9828)
  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 9828)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 24, 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: pTW334e4
FEATURES             Location/Qualifiers
     source          1..9828
                     /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             3963..3985
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             5252..5260
                     /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             5678..5700
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    7422..7438
                     /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(7446..7452,7453..7468,7469..7474))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     promoter        7717..7794
                     /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             7795..8877
                     /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(7814..7833)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      9034..9772
                     /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(9822..9828,1..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 gacggctgaa attaatgagg
     2521 agatatacat atggtatcga gaattgcgaa tcgaaagagc aaaaagctct tgattgtgaa
     2581 tgaggatgtt gctggtaatt acgacgatac cttcggtgat gtacaaaagc aaattgttaa
     2641 ttattttaca tacaaagctg tgaggacggt tcttcatcag ctgtacgaaa tgaatcctcc
     2701 tcaatacact tggttctata atcatatcat aacaaacaga ccgacggatg gcaaacgttt
     2761 cctccgtgcc ctcggcaagg agagtcagga gcttgcagaa agagtgatga tcacgcgtct
     2821 tcacttgtat ggcaaatgga tcaagaaatg cgaccatggg aagatatacc aggaaatatc
     2881 ggatgaaaac ttggcgttga tgcgtgaacg cctgatggag acagtgatat ggccttccga
     2941 tgacacaaac tcaaggtaat tcagctaaga cactgcacag gagatataca tatgatggcc
     3001 gcaaacaata atcctcaggg cactaaacca aacagtttag tttgtgcaaa ttgtgaaggg
     3061 gaaggttgtg tggcatgttc tcaatgcaaa ggaggtggtg tgaatttgat tgatcatttc
     3121 aatggtcagt ttaaagctgg tgcactctgt tggctttgca gaggtaaaaa ggaggtgttg
     3181 tgtggggact gcaatggagc tggtttcatt ggtgggttct tgagcacctt cgatgagtag
     3241 agtcagtagg gccctaaaaa aaaaccccgc ccctgacagg gcagggtttt ttttggcgaa
     3301 aaaaccccgc tgaagcgggg ttttttgcgt acattggctc cgtctaagaa agtatagcca
     3361 tcacatctga agctctgagg gcaatgtagt tggaaccatc tttgcccttg aagtcgttac
     3421 cagcatactt ggagtaaagt actgtgcttc cggttgatac tggtagaggc gtaattttac
     3481 cttcctcgtc tagggaaccc ggtccaactg ctatcacggt gccaatagaa ggcttctctt
     3541 tggtagtctc ggttaacaac aaccctccag ctgttttctc ctccgcctca gcaaccttaa
     3601 taaagactcg gtcattcaaa ggtttgagat ctttgatgtc ctctgtctca agaatgccaa
     3661 caatatcatc ttccttgagg ataagatgct tcacatcatt gaactccacc tcagttcctg
     3721 cgtatttgga gtagataatt tgtgctccag tagggacagt gatatcaatt ttgttcttcc
     3781 caatagttct tccttcaccc acagcaacga cttcacctcc ttgaggtttt gattgagcag
     3841 tggatggaag taagatacca cctaaagtct tctcctctgc ctccttgatc ttcaccaaaa
     3901 ctcgatctcc caatggctta attgaagtat acttaggggc aacaacagag gccatatgta
     3961 tatctccttc ttaaagttaa acaaaattat ttctagaggg caattgttat ccggtcgacg
     4021 tcgaccttag tatccgtaac ctgagttgtc cattgggttg ccgacgggaa ctggttcagg
     4081 ttccttgatc tcaacaacaa cgcagtccga catcaagaaa gtctttgcaa ccgaagctgc
     4141 gtgttccaag caacatctca caacctttgt tggatcgatg attcctgcag ccatcaaatc
     4201 ctcgtacttg ccggttgcag cattgtaacc gaatttcacg ttatcgttgg aaagcacctt
     4261 ctcgctaact acacttccat taacaccagc attcttggcg attagtttta gggggtaact
     4321 tagtgctctt ttaactatat ccgctccaac cttttcttca tcattatcaa gcgtggcttt
     4381 aatggcatca accttggaag caagacgaag cagagtgcaa ccaccaccaa caacaatacc
     4441 ttcctcgaca gcggcctttg ttgcattaag agcatcttca actctcaact tcttctcttt
     4501 gagttctgtc tcagtttgtg ctccaacctg aatcacagca actccaccag agagctttgc
     4561 aattctctca ttgagtttct ccttctcgta atcttgctcc gcttgctcaa taagattttt
     4621 aatctgtgta acacgctttt tcactgcatc ctgtgtgctt ccatcaccca ctatggtcga
     4681 ggtttccttt gtgaggacaa cctttgaagc attaccaaga acctcttttc cagctttgtc
     4741 aagtgaaaga ccaacttcct cacggatcac agttgctcca gttagaatgg caatatcatc
     4801 aaggtattgg ctcttgcgct ctccaaaccc tggagctctg agagctgcta tcttcagtgt
     4861 acctctaagc ttgtttacaa caagagtcgc taaagcttct tgttcaatgt cttcagcaat
     4921 aattaatatt gggtatccgc ctctaattgc atcctccaaa acaccaacaa gatcccttgc
     4981 attggtaatt ttcttgtcaa cgagaagtaa cttgcaattg tcgaactcga cggacatttt
     5041 ctcgctgtct gtcacaaagt aaggagagat ataaccacgg tcaaattgca ttccttctac
     5101 gacgtagagg ttgttctcgg cacttttacc ctcctcaagg gtaaccacac ctttcctgcc
     5161 cactttgctc attgcttcag caatcatatt tccaatttcg tcgttgttac ccgcactaac
     5221 agctgcaaca tctgcaagtt cactgtcttc aacctcctta gacattttct tgagctcggt
     5281 aaccaaagcc tttgctgtct tctcaatacc tctggtgatc aaaacagggt ttgcaccagc
     5341 agctaccacc ttgacaccct cagcgataaa accttgtgca agaacaacag atgttgttgt
     5401 accgtcacca gccaagtcat tggttttggc agccgcttgc ctcacaagct tagcgccaat
     5461 gttctcaacc gggtcttcca actcaacctc tcttgcaaca gtcacaccat cattaacaat
     5521 tcgtggtgat ccatacttgc tctcaagaac aacatttcgt cctttgggtc caagtgtaac
     5581 gccaacaaga tctgcgagct tgttgacacc agcttgaagt ctcctaatgg tagtcccatc
     5641 tttgttgaaa tgtaattcct ttgctgccat atgtatatct ccttcttaaa gttaaacaaa
     5701 attatttcta gctccttaca ccatgaggcc ctcaggagca gcagcagcag gagccttggg
     5761 tttcggtttg tcaacaacaa tggcctgagt ggtcagtacc attcctgcta cggaagcagc
     5821 gttctgtagc gcacatcttg tcactttagc cggatcaatc actccagctt cgaacagatt
     5881 ctcgtaagta tcagtcattg cgttgtaccc attctcccaa tcgctgaaca taatcttttc
     5941 cacaacaact tctccttcaa ctccagcgtt ctgagcaata agagcagctg gtgacagcaa
     6001 tgccttttgt actatgtcag ctcccaaacg ttcatcagca tcctcaaaag tctccttaat
     6061 ggcgggaata acagtggaga gatgcaccaa agcagcacca ccacctggaa ctatgccttc
     6121 ctcgatagca gcaaatgttg cgttctttgc atcctcgata cgaagcttac ggtcctcaag
     6181 ctcagtttca gttgctgctc cgactttaat gacagcaaca cctccagata gcttcgcaat
     6241 tctctcagcg agcttctctg agtcatacac agaatcagtc tcaaatagtt ctttcttcag
     6301 ctgagcaatc cgagcttgta attcgtcctt ggaagctgca tctgcaatga gggtagtcga
     6361 atctttgcta atcgtgactt ttcgggcaat acccaattga tctatggttg cgttttcaac
     6421 cagtaagctc atgtccatgg ctaggtactc agctcctgtc aagattgcaa tatcttgaag
     6481 catggctttt cttctttctc caaatcctgg cgctttaacg gcaacaacat tgaggacacc
     6541 acgaagcttg ttcacgacaa gggtagctaa ggcttcacca gtaacatcct ctgcaataat
     6601 caacaacgga gcacgaagct gagtggtctt ctccaaaata gggatgatgt ctttgatcgc
     6661 agtgatcttc tgatcagtga tcaataccct agcattctca aactcagcta gtagtttctc
     6721 agggtttgta acaaactgag gcgagatgta acctctgtca atctccatcc cttcttcaac
     6781 ttcgaccgtg gtttcaaaag aagatgaaga ttcaatggac aaaacaccat caggtccaac
     6841 tttgtcgatg gcatcagcaa tcattgaccc tataaggtca tcatttccag cagagataga
     6901 agccacagct ctgatgtcat cacgaccttt cacgggccta gctttcttct gaagctcttc
     6961 gatcagacct tgaacagtct tatcaattcc cctcttgagt gaaacgggat tcgcaccaga
     7021 agtgacgctc aacaatccat gtttgattat ctcccgagca aggatagacg cagtggttgt
     7081 cccatcacca gcagagtcat tagtcttact cgcaacctca cgaataagcg ctgcaccagc
     7141 attttccatg gcattaggta actcaatagc tctagcaatg gtgactccat cattcacaac
     7201 cttaggactt ccaaattcat ccaacacaac attcctccct ctggggccaa gcgtaagacc
     7261 aacacaatca gcaagcttat caataccagc ttgtagagca gctctagagt gctggtcaaa
     7321 agctatttcc tttacattca tatgtatatc tcctactgca cctattgttg gtgaagtgct
     7381 cgtgaaaaca cctaaacgga ctggctgttt cctgtgtgaa attgttatcc gctcacaatt
     7441 ccacacatta tacgagccga tgattaattg tcaacagctc atttcagaat atttgccaga
     7501 accgttatga tgtcggcgca aaaaacatta tccagaacgg gagtgcgcct tgagcgacac
     7561 gaattatgca gtgatttacg acctgcacag ccataccaca gcttccgatg gctgcctgac
     7621 gccagaagca ttggtgcacc gtgcagtcga tgataagctg tcaaacatga gaattgtgcc
     7681 taatgagtga gctaacttac attaattgcg ttgcgcgaca ccatcgaatg gtgcaaaacc
     7741 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     7801 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     7861 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     7921 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     7981 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     8041 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     8101 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     8161 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     8221 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     8281 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     8341 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     8401 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     8461 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     8521 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     8581 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     8641 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     8701 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     8761 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     8821 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgaagg
     8881 gtccctaagt ctcctcagca aaacgaaagg cccagtcttt cgactgagcc tttcgtttta
     8941 tttgaccgga tgtcctcttc ctcctgggct gacttcaggt gctacatttg aagagataaa
     9001 ttgcactgaa atctagagcg gttcagtaga aaagatcaaa ggatcttctt gagatccttt
     9061 ttttctgcgc gtaatctttt gccctgtaaa cgaaaaaacc acctggggag gtggtttgat
     9121 cgaaggttaa gtcagttggg gaactgctta accgtggtaa ctggctttcg cagagcacag
     9181 caaccaaatc tgtccttcca gtgtagccgg actttggcgc acacttcaag agcaaccgcg
     9241 tgtttagcta aacaaatcct ctgcgaactc ccagttacca atggctgctg ccagtggcgt
     9301 tttaccgtgc ttttccgggt tggactcaag tgaacagtta ccggataagg cgcagcagtc
     9361 gggctgaacg gggagttctt gcttacagcc cagcttggag cgaacgacct acaccgagcc
     9421 gagataccag tgtgtgagct atgagaaagc gccacacttc ccgtaaggga gaaaggcgga
     9481 acaggtatcc ggtaaacggc agggtcggaa caggagagcg caagagggag cgacccgccg
     9541 gaaacggtgg ggatctttaa gtcctgtcgg gtttcgcccg tactgtcaga ttcatggttg
     9601 agcctcacgg ctcccacaga tgcaccggaa aagcgtctgt ttatgtgaac tctggcagga
     9661 gggcggagcc tatggaaaaa cgccaccggc gcggccctgc tgttttgcct cacatgttag
     9721 tcccctgctt atccacggaa tctgtgggta actttgtatg tgtccgcagc gcccgccgca
     9781 gtctcacgcc cggagcgtag cgaccgagtg acggccttaa tttggctt
//
