LOCUS       sequence-467654-        6972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing and purifying R.rubrum rubisco using a his-sumo tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6972)
  AUTHORS   Prywes N, Phillips NR, Oltrogge LM, Lindner S, Taylor-Kearney LJ, 
            Tsai YC, de Pins B, Cowan AE, Chang HA, Wang RZ, Hall LN, 
            Bellieny-Rabelo D, Nisonoff HM, Weissman RF, Flamholz AI, Ding D, 
            Bhatt AY, Mueller-Cajar O, Shih PM, Milo R, Savage DF
  TITLE     A map of the rubisco biochemical landscape.
  JOURNAL   Nature. 2025 Jan 22. doi: 10.1038/s41586-024-08455-0.
  PUBMED    39843747
REFERENCE   2  (bases 1 to 6972)
  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: "Nature. 
            2025 Jan 22. doi: 10.1038/s41586-024-08455-0."
COMMENT     Sequence Label: NP-11-63
FEATURES             Location/Qualifiers
     source          1..6972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(351..369)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      365..412
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      449..904
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(536..555)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     746..767
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(996..1811)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(996..1811)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1719..1738
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1803..1822
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1933..2521
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2422..2441
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2675..2692
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2707..2846
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2772..2791)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2932..2954
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2948..3139)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRS*TLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3948..5030)
                     /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     4992..5011
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5031..5108)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5314..5333
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5417..5436
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5417..5435
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5436..5460
                     /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             5475..5497
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tggcggaatg aatgccttac gcatgcccgg cttcttcgaa aatcttggta atgcgaatgt
       61 catcctgact gctggtggcg gtgctttcgg ccatattgac ggccctgtgg ccggcgctcg
      121 ttctcttcgc caagcttggc aggcgtggcg cgatggcgtc cctgtgcttg actacgcccg
      181 cgagcataag gagttggcac gtgcttttga gtccttccct ggcgacgctg accagattta
      241 tccaggttgg cgtaaggcgc ttggagtgga ggatactcgt tcagctttac ctgcgtaatg
      301 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
      361 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
      421 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
      481 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
      541 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
      601 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
      661 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
      721 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
      781 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
      841 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
      901 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      961 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     1021 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     1081 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     1141 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     1201 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     1261 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     1321 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     1381 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     1441 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     1501 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     1561 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     1621 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     1681 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     1741 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     1801 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     1861 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1921 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1981 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2041 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     2101 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2161 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2221 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2281 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2341 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2401 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2461 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2521 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     2581 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     2641 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     2701 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     2761 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     2821 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     2881 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2941 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     3001 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     3061 tgagtttctc cagaagcgtt aatgtctagc ttctgataaa gcgggccatg ttaagggcgg
     3121 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     3181 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     3241 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     3301 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     3361 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     3421 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     3481 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     3541 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     3601 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     3661 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     3721 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     3781 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     3841 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     3901 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     3961 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4021 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     4081 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     4141 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     4201 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     4261 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     4321 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     4381 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     4441 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     4501 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     4561 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     4621 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     4681 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     4741 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     4801 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     4861 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     4921 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     4981 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     5041 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     5101 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     5161 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     5221 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     5281 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     5341 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     5401 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     5461 cctctagaaa taattttgtt taactttaag aaggagatat accatgagca agcatcacca
     5521 tcattcaggc catcaccata ccggacacca ccatcattca ggcagtcatc accattccgg
     5581 atctgctgcg ggtggcgaag aagataagaa accggcaggt ggcgaaggtg gcggtgccca
     5641 tatcaacctg aaagtgaaag gtcaagacgg caacgaagtc tttttccgca tcaaacgttc
     5701 tacccagctg aaaaagctga tgaacgcata ctgtgaccgt cagtctgtag acatgaccgc
     5761 aattgctttc ctgtttgatg gtcgtcgcct gcgtgcggaa cagaccccgg atgaactgga
     5821 gatggaagat ggcgacgaaa tcgacgcaat gcttcatcag actggtggca tggaccagag
     5881 ttctcgctat gtcaatcttg ctttgaaaga agaggactta attgccggag gcgaacacgt
     5941 attgtgcgcc tatattatga aaccaaaagc tgggtacggt tacgttgcca ccgcagcgca
     6001 ctttgctgca gagagttcga ctggaacaaa tgtagaggtg tgtaccactg atgattttac
     6061 tcgtggcgtg gatgccttag tttacgaagt agatgaggcc cgcgagctta ctaagattgc
     6121 ctacccagta gcactgttcg atcgtaatat cacggatgga aaagctatga tcgcatcatt
     6181 tcttacattg accatgggca ataatcaagg catgggtgac gtagaatatg ccaaaatgca
     6241 tgacttctat gtgcctgaag cgtatcgcgc tctgtttgat ggcccttctg taaatatttc
     6301 cgccttgtgg aaggtactgg gtcgtccgga ggttgatgga ggcttagttg tcggtactat
     6361 cattaaacca aagcttgggc tgcgccctaa gcctttcgct gaggcttgcc acgcattttg
     6421 gttaggtggt gattttatta aaaatgatga accgcaaggg aaccaaccct tcgcgccact
     6481 tcgcgacacg atcgccttgg ttgcagatgc gatgcgccgt gcacaggacg aaacgggtga
     6541 agcaaagttg tttagtgcta atatcaccgc tgatgacccg tttgaaatca tcgcccgcgg
     6601 ggaatacgtt cttgaaacat tcggtgaaaa tgcaagtcac gttgccttat tagtagacgg
     6661 atacgtagcc ggcgcagctg ctattacgac cgctcgtcgc cgtttccccg ataatttctt
     6721 gcattatcat cgcgccgggc atggagcggt cactagccct cagtctaagc gcggctatac
     6781 tgcttttgtg cattgtaaga tggctcgttt gcaaggtgcg tcgggtatcc ataccggaac
     6841 tatgggcttt ggaaagatgg aaggggagtc ctcagatcgt gccatcgcgt atatgttaac
     6901 tcaagatgaa gcacaaggcc ccttttatcg ccagtcgtgg ggagggatga aggcctgcac
     6961 ccctattatt tc
//
