LOCUS       sequence-472478-        7501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses M. truncatula RbcL RbcS-6xHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7501)
  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 7501)
  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: pTW335m
FEATURES             Location/Qualifiers
     source          1..7501
                     /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             5175..5197
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5580..5597
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5679..5726
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5960..5978)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6046..6150
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6151..6219,6220..7011)
                     /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(6369..6388)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7182..7501,1..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 taccatgtca ccacaaacag agactaaagc aacggtaggt tttaaggctg
     3781 gcgtgaagga ttatcgtctt acttactaca cgccggacta cgagactaaa gacacagata
     3841 ttctagccgc atttcgtgtg tcaccgcaac caggtgtgcc ggccgaagag gcgggagccg
     3901 ctgtggccgc cgaaagctcc accggcactt ggacaactgt gtggaccgat ggtctgacca
     3961 gcctcgatcg ctacaaaggt cgttgttatc atattgagcc tgtggcgggt gaagaaagcc
     4021 aattcatcgc atatgtagct tatccgctgg atctgtttga ggaaggcagc gtgaccaata
     4081 tgttcacgtc cattgtaggt aatgtctttg gctttaaagc gcttcgtgcg ttgcgtctgg
     4141 aagatctgcg cataccagtg gcgtatgtaa aaaccttcca agggccgccc cacggtatcc
     4201 aggttgaacg cgacaaactg aacaaatacg gtcgaccgct cctcggatgc accataaagc
     4261 cgaaattagg cctgagtgca aaaaattatg gccgcgccgt gtacgagtgc ctgcgggggg
     4321 gtctggattt caccaaggac gatgaaaacg tcaattctca gccctttatg cgctggcgtg
     4381 atcgttttct gttctgtgcg gaagctattt ataaggcgca ggccgaaacc ggcgaaatca
     4441 aaggccacta tctgaacgcg accgccggca cgtgcgagga tatgatgaaa agagcggttt
     4501 ttgcgcgcga actgggcgtt cctatcgtaa tgcatgatta cttaacaggg ggctttacgg
     4561 ctaacacaac tctggcacac tactgtcgag ataatggcct gctgctgcat attcatcgtg
     4621 caatgcatgc ggtcattgat cgtcagaaaa accatggaat gcactttcgt gtgcttgcca
     4681 aagcccttcg tatgtcaggc ggcgatcata ttcacgcagg caccgtcgtt ggcaaattag
     4741 aaggtgagcg cgatattacg ctaggctttg tggatttgtt aagggacgac ttcgtggaga
     4801 aagaccggtc gcgcggcatt ttcttcaccc aggactgggt gagtctgcct ggcgtattgc
     4861 cagttgcgag cggtgggatc catgtttggc acatgccggc gcttacagag atttttggtg
     4921 atgactcggt cttacagttc ggaggtggca ccttgggcca tccttggggg aacgcaccgg
     4981 gcgcggttgc aaatcgcgtt gccctggaag cgtgcgtgca ggcacgcaat gaaggacgcg
     5041 acctcgcgcg cgaagggaac gaaatcatcc gcgaagccac caaatggagc ccggaactag
     5101 ctgctgcgtg cgaagtctgg aaagagatta agttcgaatt tccggcgatg gataccatct
     5161 aagctagaaa taattttgtt taactttaag aaggagatat acatatgtgt atgcaagttt
     5221 ggccccccat tggaaaaaag aagtttgaga ctctgtcgta cctgcctccg ctcaccgaag
     5281 aacagttagc gaaagaagtt gaatacctga ttcgaaaagg ctgggtgccg tgcctagaat
     5341 ttgagctgga aaaaggcttc gtataccgcg agaaccactc aagcccgggg tattacgatg
     5401 gtcgctattg gactatgtgg aaactgcctc tttttggcgc cacagacagc tcccaggtgt
     5461 taaaagaatt ggcggaggct aaagcagcat atccggaatc tttcatccgt ataattggtt
     5521 tcgataatgt gcgtcaggtt caatgtatta gttttatcgc ccataccccc gcgacgtatc
     5581 atcaccacca tcatcattaa catccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     5641 ctgctgccac cgctgagcaa taaaccgctg agcaataact agcataaccc cttggggcct
     5701 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa
     5761 gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc
     5821 gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt
     5881 taactgtgat aaactacagc attaaagcta gcttatcgat gataagctgt caaacatgag
     5941 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     6001 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6061 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6121 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6181 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6241 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6301 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6361 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     6421 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6481 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6541 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6601 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6661 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     6721 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6781 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6841 gataaatctg gagccggtga gcgtggatct cgcggtatca ttgcagcact ggggccagat
     6901 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6961 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7021 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7081 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7141 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7201 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7261 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7321 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7381 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7441 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7501 a
//
