LOCUS       sequence-472473-        7512 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses N. tabacum RbcL RbcS-6xHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7512)
  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 7512)
  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: pTW335i3
FEATURES             Location/Qualifiers
     source          1..7512
                     /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             5592..5609
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5690..5737
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5971..5989)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6057..6161
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6162..6230,6231..7022)
                     /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(6380..6399)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7193..7512,1..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 acgtcagcaa 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 tgttggattc aaagctggtg
     3781 ttaaagagta caaattgact tattatactc ctgagtacca aaccaaggat actgatatat
     3841 tggcagcatt ccgagtaact cctcaacctg gagttccacc tgaagaagca ggggccgcgg
     3901 tagctgccga atcttctact ggtacatgga caactgtatg gaccgatgga cttaccagcc
     3961 ttgatcgtta caaagggcga tgctaccgca tcgagcgtgt tgttggagaa aaagatcaat
     4021 atattgctta tgtagcttac cctttagacc tttttgaaga aggttctgtt accaacatgt
     4081 ttacttccat tgtaggtaac gtatttgggt tcaaagccct gcgcgctcta cgtctggaag
     4141 atctgcgaat ccctcctgct tatgttaaaa ctttccaagg tccgcctcat gggatccaag
     4201 ttgaaagaga taaattgaac aagtatggtc gtcccctgtt gggatgtact attaaaccta
     4261 aattggggtt atctgctaaa aactacggta gagctgttta tgaatgtctt cgcggtggac
     4321 ttgattttac caaagatgat gagaacgtga actcacaacc atttatgcgt tggagagatc
     4381 gtttcttatt ttgtgccgaa gcactttata aagcacaggc tgaaacaggt gaaatcaaag
     4441 ggcattactt gaatgctact gcaggtacat gcgaagaaat gatcaaaaga gctgtatttg
     4501 ctagagaatt gggcgttccg atcgtaatgc atgactactt aacgggggga ttcaccgcaa
     4561 atactagctt ggctcattat tgccgagata atggtctact tcttcacatc caccgtgcaa
     4621 tgcatgcggt tattgataga cagaagaatc atggtatcca cttccgggta ttagcaaaag
     4681 cgttacgtat gtctggtgga gatcatattc actctggtac cgtagtaggt aaacttgaag
     4741 gtgaaagaga cataactttg ggctttgttg atttactgcg tgatgatttt gttgaacaag
     4801 atcgaagtcg cggtatttat ttcactcaag attgggtctc tttaccaggt gttctacccg
     4861 tggcttcagg aggtattcac gtttggcata tgcctgctct gaccgagatc tttggggatg
     4921 attccgtact acagttcggt ggaggaactt taggacatcc ttggggtaat gcgccaggtg
     4981 ccgtagctaa tcgagtagct ctagaagcat gtgtaaaagc tcgtaatgaa ggacgtgatc
     5041 ttgctcagga aggtaatgaa attattcgcg aggcttgcaa atggagcccg gaactagctg
     5101 ctgcttgtga agtatggaaa gagatcgtat ttaattttgc agcagtggac gttttggata
     5161 agtaagctag aaataatttt gtttaacttt aagaaggaga tatacatatg tgtatgcaag
     5221 tttggccccc gatcaataaa aaaaaatacg aaaccctgag ctatttaccc gatctgtcgc
     5281 aggagcagct gctgagcgag gttgagtacc tcctgaagaa cggttgggtc ccatgcttag
     5341 aattcgaaac agagcatggc tttgtttatc gcgaaaacaa caaaagtccg ggctactacg
     5401 atggccgtta ttggactatg tggaagttgc ctatgttcgg gtgtacggat gcgacccagg
     5461 tacttgccga agtggaagaa gctaaaaaag catatccgca ggcgtggatt cgcatcattg
     5521 gatttgacaa tgtccgtcag gtgcaatgca tttcatttat tgcctacaaa ccggaaggtt
     5581 atgcactcga gcatcaccac catcatcatt aaatccggct gctaacaaag cccgaaagga
     5641 agctgagttg gctgctgcca ccgctgagca ataaaccgct gagcaataac tagcataacc
     5701 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     5761 atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg
     5821 gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg
     5881 cgttagcaat ttaactgtga taaactacag cattaaagct agcttatcga tgataagctg
     5941 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     6001 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     6061 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     6121 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     6181 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     6241 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     6301 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     6361 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     6421 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     6481 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     6541 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     6601 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6661 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     6721 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6781 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6841 ggtttattgc tgataaatct ggagccggtg agcgtggatc tcgcggtatc attgcagcac
     6901 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6961 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     7021 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     7081 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     7141 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     7201 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     7261 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     7321 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     7381 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     7441 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     7501 ggtcgggctg aa
//
