LOCUS       sequence-472501-        7527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses O. sativa RbcL RbcS-6xHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7527)
  AUTHORS   Archer J, Kathpalia M, Lee BU, Li S, Wang T
  TITLE     Effects of chaperone selectivity on the assembly of plant Rubisco 
            orthologs in E. coli.
  JOURNAL   J Exp Bot. 2025 Mar 31:eraf140. doi: 10.1093/jxb/eraf140.
  PUBMED    40163664
REFERENCE   2  (bases 1 to 7527)
  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: "J Exp Bot. 
            2025 Mar 31:eraf140. doi: 10.1093/jxb/eraf140."
COMMENT     Sequence Label: pTW335h3
FEATURES             Location/Qualifiers
     source          1..7527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     170..189
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     423..440
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    455..594
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(520..539)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     680..702
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(696..887)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2199..3281)
                     /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     3243..3262
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3282..3359)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(3602..3607,3608..3623,3624..3630)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3638..3654
                     /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)."
     primer_bind     3643..3665
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     RBS             5178..5200
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5607..5624
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5705..5752
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5986..6004)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6072..6176
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6177..6245,6246..7037)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6395..6414)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7208..7527,1..269)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
       61 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      121 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
      181 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
      241 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
      301 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
      361 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
      421 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
      481 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
      541 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
      601 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
      661 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
      721 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
      781 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
      841 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
      901 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
      961 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     1021 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     1081 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     1141 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     1201 accattcatg ttgttgctca ggtcgcagat gttttgcagc agcagtcgct tcacgttcgc
     1261 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     1321 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
     1381 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
     1441 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
     1501 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     1561 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
     1621 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
     1681 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
     1741 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     1801 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     1861 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     1921 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     1981 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     2041 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     2101 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     2161 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2221 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2281 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     2341 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     2401 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     2461 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     2521 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     2581 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     2641 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     2701 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     2761 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     2821 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     2881 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     2941 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     3001 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     3061 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     3121 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     3181 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     3241 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     3301 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcg
     3361 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac aattctcatg tttgacagct
     3421 tatcatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
     3481 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
     3541 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
     3601 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
     3661 acaggaaaca gccagtccgt ttaggtgttt tcacgagcac ttcaccaaca ataggtgcag
     3721 taaggagata taccatgcca caaacagaga ctaaagcaag tgttggattt aaagctggtg
     3781 ttaaggatta taaattgact tactacaccc cggagtacga aaccaaggac actgatatct
     3841 tggcagcatt ccgagtaact cctcagccgg gggttccgcc cgaagaagca ggggctgcag
     3901 tagctgccga atcttctact ggtacatgga caactgtttg gactgatgga cttaccagtc
     3961 ttgatcgtta caaaggccga tgctatcaca tcgagcccgt tgttggggag gataatcaat
     4021 atatcgctta tgtagcttat ccattagacc tatttgaaga gggttctgtt actaacatgt
     4081 ttacttccat tgtgggtaac gtatttggtt tcaaagccct acgcgctcta cgtctggagg
     4141 atctgcgaat tccccctact tattcaaaaa ctttccaagg tccgcctcat ggtatccaag
     4201 ttgaaaggga taagttgaac aaatacggtc gtcctttatt gggatgtact attaaaccaa
     4261 aattgggatt atctgcaaaa aattatggta gagcatgtta tgagtgtcta cgcggtggac
     4321 ttgattttac caaagatgat gaaaacgtaa actcacaacc atttatgcgt tggagggacc
     4381 gttttgtctt ttgtgccgaa gctatttata aatcacaggc cgaaaccggt gaaattaagg
     4441 ggcattactt gaatgcgact gcaggtacat gcgaagaaat gattaaaaga gctgtatttg
     4501 cgagggaatt aggggttcct attgtaatgc atgactactt aaccgggggg ttcaccgcaa
     4561 atactagttt ggctcattat tgccgcgaca acggcctact tcttcacatt caccgagcaa
     4621 tgcatgcagt tattgataga cagaaaaatc atggtatgca tttccgtgta ttagctaaag
     4681 cattgcgtat gtctggggga gatcatatcc acgctggtac agtagtaggt aagttagaag
     4741 gggaacgcga aatgacttta ggttttgttg atttattgcg cgatgatttt attgaaaaag
     4801 atcgtgctcg cggtatcttt ttcactcagg actgggtatc catgccaggt gttataccgg
     4861 tggcttcagg gggtattcat gtttggcata tgccagctct gaccgaaatc tttggagatg
     4921 attctgtatt gcaatttggt ggaggaactt taggacatcc ttggggtaat gcacctggtg
     4981 cagcagctaa tcgggtggct ttagaagcct gtgtacaagc tcgtaacgaa gggcgcgatc
     5041 ttgctcgtga aggtaatgaa attatccgat cagcttgcaa atggagtcct gaactagccg
     5101 cagcttgtga aatatggaaa gcgatcaaat tcgagttcga gccggtagat aaactagata
     5161 gctaggctag aaataatttt gtttaacttt aagaaggaga tatacatatg tgtatgcaag
     5221 tttggcccat tgaaggtata aaaaaatttg aaaccctgtc atacttaccc ccgctgacgg
     5281 tcgaagacct tctaaagcaa atcgagtatc tgctccgatc gaagtgggtt ccatgcctgg
     5341 aattcagtaa agtgggtttt gtataccgtg aaaaccatcg tagcccgggg tattacgatg
     5401 gccgctattg gactatgtgg aagctgccga tgttcggctg taccgatgcc acacaggtgt
     5461 tgaaagagct ggaggaagcc aaaaaagcgt acccggatgc gttcgttcgc attatcggct
     5521 ttgacaacgt gcgccaggtc caattaattt cctttattgc atataaacct cctggctgcg
     5581 aagaaagcgg aggtaatgca ctcgagcatc accaccatca tcattaaatc cggctgctaa
     5641 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataaa ccgctgagca
     5701 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     5761 aggaactata tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg
     5821 cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata
     5881 cacggtgcct gactgcgtta gcaatttaac tgtgataaac tacagcatta aagctagctt
     5941 atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc
     6001 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     6061 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6121 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6181 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6241 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6301 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6361 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
     6421 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6481 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6541 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6601 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6661 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6721 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6781 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6841 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggatctcgcg
     6901 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6961 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     7021 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     7081 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     7141 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     7201 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     7261 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     7321 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     7381 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     7441 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7501 cggataaggc gcagcggtcg ggctgaa
//
