LOCUS       sequence-490606-       10091 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 10091)
  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 10091)
  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: pTW334e2
FEATURES             Location/Qualifiers
     source          1..10091
                     /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             4226..4248
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             5515..5523
                     /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             5941..5963
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    7685..7701
                     /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(7709..7715,7716..7731,7732..7737))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     promoter        7980..8057
                     /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             8058..9140
                     /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(8077..8096)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      9297..10035
                     /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(10085..10091,1..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 gcgtgacggc tgaaattaat
     3061 gaggagatat acatatggta tcgagaattg cgaatcgaaa gagcaaaaag ctcttgattg
     3121 tgaatgagga tgttgctggt aattacgacg ataccttcgg tgatgtacaa aagcaaattg
     3181 ttaattattt tacatacaaa gctgtgagga cggttcttca tcagctgtac gaaatgaatc
     3241 ctcctcaata cacttggttc tataatcata tcataacaaa cagaccgacg gatggcaaac
     3301 gtttcctccg tgccctcggc aaggagagtc aggagcttgc agaaagagtg atgatcacgc
     3361 gtcttcactt gtatggcaaa tggatcaaga aatgcgacca tgggaagata taccaggaaa
     3421 tatcggatga aaacttggcg ttgatgcgtg aacgcctgat ggagacagtg atatggcctt
     3481 ccgatgacac aaactcaagg tagagtcagt agggccctaa aaaaaaaccc cgcccctgac
     3541 agggcagggt tttttttggc gaaaaaaccc cgctgaagcg gggttttttg cgtacattgg
     3601 ctccgtctaa gaaagtatag ccatcacatc tgaagctctg agggcaatgt agttggaacc
     3661 atctttgccc ttgaagtcgt taccagcata cttggagtaa agtactgtgc ttccggttga
     3721 tactggtaga ggcgtaattt taccttcctc gtctagggaa cccggtccaa ctgctatcac
     3781 ggtgccaata gaaggcttct ctttggtagt ctcggttaac aacaaccctc cagctgtttt
     3841 ctcctccgcc tcagcaacct taataaagac tcggtcattc aaaggtttga gatctttgat
     3901 gtcctctgtc tcaagaatgc caacaatatc atcttccttg aggataagat gcttcacatc
     3961 attgaactcc acctcagttc ctgcgtattt ggagtagata atttgtgctc cagtagggac
     4021 agtgatatca attttgttct tcccaatagt tcttccttca cccacagcaa cgacttcacc
     4081 tccttgaggt tttgattgag cagtggatgg aagtaagata ccacctaaag tcttctcctc
     4141 tgcctccttg atcttcacca aaactcgatc tcccaatggc ttaattgaag tatacttagg
     4201 ggcaacaaca gaggccatat gtatatctcc ttcttaaagt taaacaaaat tatttctaga
     4261 gggcaattgt tatccggtcg acgtcgacct tagtatccgt aacctgagtt gtccattggg
     4321 ttgccgacgg gaactggttc aggttccttg atctcaacaa caacgcagtc cgacatcaag
     4381 aaagtctttg caaccgaagc tgcgtgttcc aagcaacatc tcacaacctt tgttggatcg
     4441 atgattcctg cagccatcaa atcctcgtac ttgccggttg cagcattgta accgaatttc
     4501 acgttatcgt tggaaagcac cttctcgcta actacacttc cattaacacc agcattcttg
     4561 gcgattagtt ttagggggta acttagtgct cttttaacta tatccgctcc aaccttttct
     4621 tcatcattat caagcgtggc tttaatggca tcaaccttgg aagcaagacg aagcagagtg
     4681 caaccaccac caacaacaat accttcctcg acagcggcct ttgttgcatt aagagcatct
     4741 tcaactctca acttcttctc tttgagttct gtctcagttt gtgctccaac ctgaatcaca
     4801 gcaactccac cagagagctt tgcaattctc tcattgagtt tctccttctc gtaatcttgc
     4861 tccgcttgct caataagatt tttaatctgt gtaacacgct ttttcactgc atcctgtgtg
     4921 cttccatcac ccactatggt cgaggtttcc tttgtgagga caacctttga agcattacca
     4981 agaacctctt ttccagcttt gtcaagtgaa agaccaactt cctcacggat cacagttgct
     5041 ccagttagaa tggcaatatc atcaaggtat tggctcttgc gctctccaaa ccctggagct
     5101 ctgagagctg ctatcttcag tgtacctcta agcttgttta caacaagagt cgctaaagct
     5161 tcttgttcaa tgtcttcagc aataattaat attgggtatc cgcctctaat tgcatcctcc
     5221 aaaacaccaa caagatccct tgcattggta attttcttgt caacgagaag taacttgcaa
     5281 ttgtcgaact cgacggacat tttctcgctg tctgtcacaa agtaaggaga gatataacca
     5341 cggtcaaatt gcattccttc tacgacgtag aggttgttct cggcactttt accctcctca
     5401 agggtaacca cacctttcct gcccactttg ctcattgctt cagcaatcat atttccaatt
     5461 tcgtcgttgt tacccgcact aacagctgca acatctgcaa gttcactgtc ttcaacctcc
     5521 ttagacattt tcttgagctc ggtaaccaaa gcctttgctg tcttctcaat acctctggtg
     5581 atcaaaacag ggtttgcacc agcagctacc accttgacac cctcagcgat aaaaccttgt
     5641 gcaagaacaa cagatgttgt tgtaccgtca ccagccaagt cattggtttt ggcagccgct
     5701 tgcctcacaa gcttagcgcc aatgttctca accgggtctt ccaactcaac ctctcttgca
     5761 acagtcacac catcattaac aattcgtggt gatccatact tgctctcaag aacaacattt
     5821 cgtcctttgg gtccaagtgt aacgccaaca agatctgcga gcttgttgac accagcttga
     5881 agtctcctaa tggtagtccc atctttgttg aaatgtaatt cctttgctgc catatgtata
     5941 tctccttctt aaagttaaac aaaattattt ctagctcctt acaccatgag gccctcagga
     6001 gcagcagcag caggagcctt gggtttcggt ttgtcaacaa caatggcctg agtggtcagt
     6061 accattcctg ctacggaagc agcgttctgt agcgcacatc ttgtcacttt agccggatca
     6121 atcactccag cttcgaacag attctcgtaa gtatcagtca ttgcgttgta cccattctcc
     6181 caatcgctga acataatctt ttccacaaca acttctcctt caactccagc gttctgagca
     6241 ataagagcag ctggtgacag caatgccttt tgtactatgt cagctcccaa acgttcatca
     6301 gcatcctcaa aagtctcctt aatggcggga ataacagtgg agagatgcac caaagcagca
     6361 ccaccacctg gaactatgcc ttcctcgata gcagcaaatg ttgcgttctt tgcatcctcg
     6421 atacgaagct tacggtcctc aagctcagtt tcagttgctg ctccgacttt aatgacagca
     6481 acacctccag atagcttcgc aattctctca gcgagcttct ctgagtcata cacagaatca
     6541 gtctcaaata gttctttctt cagctgagca atccgagctt gtaattcgtc cttggaagct
     6601 gcatctgcaa tgagggtagt cgaatctttg ctaatcgtga cttttcgggc aatacccaat
     6661 tgatctatgg ttgcgttttc aaccagtaag ctcatgtcca tggctaggta ctcagctcct
     6721 gtcaagattg caatatcttg aagcatggct tttcttcttt ctccaaatcc tggcgcttta
     6781 acggcaacaa cattgaggac accacgaagc ttgttcacga caagggtagc taaggcttca
     6841 ccagtaacat cctctgcaat aatcaacaac ggagcacgaa gctgagtggt cttctccaaa
     6901 atagggatga tgtctttgat cgcagtgatc ttctgatcag tgatcaatac cctagcattc
     6961 tcaaactcag ctagtagttt ctcagggttt gtaacaaact gaggcgagat gtaacctctg
     7021 tcaatctcca tcccttcttc aacttcgacc gtggtttcaa aagaagatga agattcaatg
     7081 gacaaaacac catcaggtcc aactttgtcg atggcatcag caatcattga ccctataagg
     7141 tcatcatttc cagcagagat agaagccaca gctctgatgt catcacgacc tttcacgggc
     7201 ctagctttct tctgaagctc ttcgatcaga ccttgaacag tcttatcaat tcccctcttg
     7261 agtgaaacgg gattcgcacc agaagtgacg ctcaacaatc catgtttgat tatctcccga
     7321 gcaaggatag acgcagtggt tgtcccatca ccagcagagt cattagtctt actcgcaacc
     7381 tcacgaataa gcgctgcacc agcattttcc atggcattag gtaactcaat agctctagca
     7441 atggtgactc catcattcac aaccttagga cttccaaatt catccaacac aacattcctc
     7501 cctctggggc caagcgtaag accaacacaa tcagcaagct tatcaatacc agcttgtaga
     7561 gcagctctag agtgctggtc aaaagctatt tcctttacat tcatatgtat atctcctact
     7621 gcacctattg ttggtgaagt gctcgtgaaa acacctaaac ggactggctg tttcctgtgt
     7681 gaaattgtta tccgctcaca attccacaca ttatacgagc cgatgattaa ttgtcaacag
     7741 ctcatttcag aatatttgcc agaaccgtta tgatgtcggc gcaaaaaaca ttatccagaa
     7801 cgggagtgcg ccttgagcga cacgaattat gcagtgattt acgacctgca cagccatacc
     7861 acagcttccg atggctgcct gacgccagaa gcattggtgc accgtgcagt cgatgataag
     7921 ctgtcaaaca tgagaattgt gcctaatgag tgagctaact tacattaatt gcgttgcgcg
     7981 acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     8041 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     8101 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     8161 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     8221 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     8281 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     8341 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     8401 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     8461 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     8521 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
     8581 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     8641 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     8701 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     8761 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     8821 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
     8881 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     8941 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     9001 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     9061 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     9121 gactggaaag cgggcagtga agggtcccta agtctcctca gcaaaacgaa aggcccagtc
     9181 tttcgactga gcctttcgtt ttatttgacc ggatgtcctc ttcctcctgg gctgacttca
     9241 ggtgctacat ttgaagagat aaattgcact gaaatctaga gcggttcagt agaaaagatc
     9301 aaaggatctt cttgagatcc tttttttctg cgcgtaatct tttgccctgt aaacgaaaaa
     9361 accacctggg gaggtggttt gatcgaaggt taagtcagtt ggggaactgc ttaaccgtgg
     9421 taactggctt tcgcagagca cagcaaccaa atctgtcctt ccagtgtagc cggactttgg
     9481 cgcacacttc aagagcaacc gcgtgtttag ctaaacaaat cctctgcgaa ctcccagtta
     9541 ccaatggctg ctgccagtgg cgttttaccg tgcttttccg ggttggactc aagtgaacag
     9601 ttaccggata aggcgcagca gtcgggctga acggggagtt cttgcttaca gcccagcttg
     9661 gagcgaacga cctacaccga gccgagatac cagtgtgtga gctatgagaa agcgccacac
     9721 ttcccgtaag ggagaaaggc ggaacaggta tccggtaaac ggcagggtcg gaacaggaga
     9781 gcgcaagagg gagcgacccg ccggaaacgg tggggatctt taagtcctgt cgggtttcgc
     9841 ccgtactgtc agattcatgg ttgagcctca cggctcccac agatgcaccg gaaaagcgtc
     9901 tgtttatgtg aactctggca ggagggcgga gcctatggaa aaacgccacc ggcgcggccc
     9961 tgctgttttg cctcacatgt tagtcccctg cttatccacg gaatctgtgg gtaactttgt
    10021 atgtgtccgc agcgcccgcc gcagtctcac gcccggagcg tagcgaccga gtgacggcct
    10081 taatttggct t
//
