LOCUS       sequence-324158-        6133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses R. eutropha Form IC rubisco and S. elongatus Prk.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6133)
  AUTHORS   Flamholz AI, Dugan E, Blikstad C, Gleizer S, Ben-Nissan R, Amram S, 
            Antonovsky N, Ravishankar S, Noor E, Bar-Even A, Milo R, Savage D
  TITLE     Functional reconstitution of a bacterial CO2 concentrating mechanism
            in E. coli.
  JOURNAL   Elife. 2020 Oct 21;9. pii: 59882. doi: 10.7554/eLife.59882.
  PUBMED    33084575
REFERENCE   2  (bases 1 to 6133)
  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: "Elife."; 
            date: "2020-10-21"; volume: "9. pii: 59882. doi"; pages: " 
            10.7554/eLife.59882"
COMMENT     Sequence Label: pFE-Reu-FI-prk
FEATURES             Location/Qualifiers
     source          1..6133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(86..674)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(166..185)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(762..856)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(887..1681)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(998..1017)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1608..1627
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1860..1878
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1899..1917,1918..1923,1924..1940,1941..1946,
                     1947..1972)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1899..1917
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1924..1942
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1982..1993
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             complement(3764..3775)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3989..4006
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5056..5142
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5315..5938)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     5846..5865
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(5939..6043)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
       61 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      121 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      181 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      241 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      301 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      361 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      421 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      481 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
      541 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      601 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      661 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      721 aaaactcacg ttaagggatt ttggtcatga ctagtgcttg gattctcacc aataaaaaac
      781 gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga
      841 tctatcaaca ggagtccaag cgagctctcg aaccccagag tcccgctcag aagaactcgt
      901 caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga
      961 ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta
     1021 tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc
     1081 cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc
     1141 cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct
     1201 cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga
     1261 tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc
     1321 gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat
     1381 cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga
     1441 gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct
     1501 gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg
     1561 ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc
     1621 cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca
     1681 tgcgaaacga tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc
     1741 ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg
     1801 ccccagctgg caattccgac gtctaagaaa ccattattat catgacatta acctataaaa
     1861 ataggcgtat cacgaggccc tttcgtcttc acctcgagtc cctatcagtg atagagattg
     1921 acatccctat cagtgataga gatactgagc acatcagcag gacgcactga ccgaattcat
     1981 taaagaggag aaaggtacca tgaacgcacc tgaatcggtc caagccaagc cgcgcaagcg
     2041 ctatgacgcc ggcgtcatga agtacaagga aatggggtac tgggacggcg actacgagcc
     2101 caaggatacc gacctgctgg cgctgttccg catcacgccg caagacggcg tggatccggt
     2161 cgaggccgct gccgcggtgg ccggcgaatc ctccaccgcc acctggacgg tggtgtggac
     2221 cgaccgcctg accgcctgcg acatgtaccg cgccaaggcc taccgggtcg atccggtgcc
     2281 caacaacccc gagcagttct tctgctacgt ggcctatgac ctgtcgctgt tcgaggaagg
     2341 ctcgatcgcc aacctgaccg cgtcgatcat cggcaacgtc ttcagcttca agccgatcaa
     2401 ggccgcgcgc ctggaagaca tgcgcttccc ggtggcctat gtgaagacct tcgccggccc
     2461 ctccacgggc atcatcgtcg agcgcgagcg cctcgacaag ttcggccgcc cgctgctggg
     2521 cgcgaccacc aagcccaagc tgggactgtc gggccgcaac tacggccgcg tggtgtatga
     2581 gggcctgaag ggcgggctgg atttcatgaa ggacgacgag aacatcaact cgcagccctt
     2641 catgcactgg cgcgaccgct tcctgttcgt gatggacgcc gtcaacaagg cttcggccgc
     2701 caccggcgag gtcaagggca gctacctcaa cgtgaccgcc ggcaccatgg aagagatgta
     2761 ccggcgcgcc gagttcgcca agtcgctggg ctcggtcgtc atcatgatcg acctgatcgt
     2821 gggctggacc tgtatccagt ccatgagcaa ctggtgccgc cagaacgaca tgatcctgca
     2881 cctgcaccgc gcgggccatg gcacctacac ccggcagaag aaccacggcg tgtccttccg
     2941 cgtgatcgcc aaatggctgc gcctggccgg cgtcgaccac atgcacaccg gcaccgcggt
     3001 gggcaagctg gaaggcgatc cgctgaccgt gcagggctac tacaacgtct gccgcgatgc
     3061 gtacacccat accgacctga cgcgcgggct gttcttcgac caggactggg cttcgctgcg
     3121 caaggtgatg ccggtggcct ccggcggcat ccatgccggc cagatgcacc agctgatcca
     3181 cctctttggc gacgacgtgg tgctgcagtt cggcggcggc accatcggcc acccgcaagg
     3241 gatccaggcg ggtgctacgg ccaaccgcgt ggcgctggaa gcgatggtgc tggcgcgcaa
     3301 cgaaggccgc gacatcctca atgaagggcc ggagatcctg cgcgatgccg cgcgctggtg
     3361 cggaccgctg cgggctgcgc tcgatacctg gggcgatatc agcttcaact acacgccgac
     3421 cgacacctcg gacttcgcgc ccaccgcgtc ggtggcctga gccctggcgc gacgccacca
     3481 aggcacgacg agcgaaccca aagaacgaga ggacatcatg cgtattactc aaggcacttt
     3541 ctctttcctg cccgacctca ccgacgcgca gatcaccagc cagcttgaat actgcctgaa
     3601 ccagggctgg gcggtcggca tcgagtacac cgacgacccg catccgcgca atacctactg
     3661 ggagatgttc ggcctgccga tgttcgacct gcgcgatgcc gccggcatcc tgctggagat
     3721 caacaacgcg cgcagcacct tccccaacca ctacatccgc gtcacggcct tcgattcgac
     3781 gcatacggtg gagtcggtgg tgatgtcgtt catcgtgaat cgtccggccg acgagcccgg
     3841 cttccgcctg gtgcgccagg aagagcccgg ccgcacgatc cgctattcga tcgagagcta
     3901 cgccgtgcag gcgcgccccg agggcagccg ctactgaatc taatagaaat aattttgttt
     3961 aactttataa gcaggaccgg cggcgatgca tcatcaccat caccacagca agccagatcg
     4021 tgttgttttg atcggcgttg ccggtgactc cggttgcggc aaatcaacct tcctaaatcg
     4081 ccttgccgac ttgtttggta cggaattgat gacggtcatc tgcttggatg actatcacag
     4141 tctcgatcgc aagggccgga aggaagcagg cgtaacggct ttggatcccc gcgccaacaa
     4201 ctttgacttg atgtatgaac aggtcaaggc gttgaagaac ggcgaaacga tcatgaagcc
     4261 gatctacaac catgaaaccg gcttgatcga tccgcccgaa aaaatcgaac ccaatcgcat
     4321 cattgtgatc gagggtctgc atccgcttta cgacgagcgc gtgcgtgaac tgctcgattt
     4381 cagcgtttac ctcgacatcg atgacgaagt caaaatcgct tggaagatcc aacgcgatat
     4441 ggcagaacgc ggccactcct acgaagatgt cctcgcctcg atcgaagcgc gccgccctga
     4501 cttcaaggcc tacattgagc cccagcgtgg ccatgcggac atcgtcatcc gcgtcatgcc
     4561 gacccagcta atccccaatg acaccgagcg caaggtgctg cgggtgcagt tgatccaacg
     4621 ggaaggccgc gatggttttg agccggctta cctgttcgac gaaggttcga ccatccagtg
     4681 gacgccctgc ggtcgtaagc tgacctgctc ctatccgggc attcgcttag cctacggccc
     4741 tgacacctac tacggtcacg aagtctcagt gcttgaggtc gacggtcagt tcgagaacct
     4801 cgaagagatg atctacgtcg agggccacct cagcaagacc gacacgcagt actacggtga
     4861 gttgacccac ctgctgctac agcacaaaga ttacccgggt tcgaacaacg gcacgggtct
     4921 gttccaagtg ctgaccggcc tgaaaatgcg ggcggcctat gagcgtttga cctcccaagc
     4981 agcacccgtc gccgctagtg tctagatgga attcctgcag cccgggggat cccatggtac
     5041 gcgtgctaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     5101 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccgcc gctagcagtt
     5161 cgtagacgga aacaaacgca gaatccaagc aaaagggaat aagggcgaca cggaaatgtt
     5221 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tatgtccact
     5281 gggttcgtgc cttcatccgg atatccagga actgttaaga cccactttca catttaagtt
     5341 gtttttctaa tccgcatatg atcaattcaa ggccgaataa gaaggctggc tctgcacctt
     5401 ggtgatcaaa taattcgata gcttgtcgta ataatggcgg catactatca gtagtaggtg
     5461 tttccctttc ttctttagcg acttgatgct cttgatcttc caatacgcaa cctaaagtaa
     5521 aatgccccac agcgctgagt gcatataatg cattctctag tgaaaaacct tgttggcata
     5581 aaaaggctaa ttgattttcg agagtttcat actgtttttc tgtaggccgt gtacctaaat
     5641 gtacttttgc tccatcgcga tgacttagta aagcacatct aaaactttta gcgttattac
     5701 gtaaaaaatc ttgccagctt tccccttcta aagggcaaaa gtgagtatgg tgcctatcta
     5761 acatctcaat ggctaaggcg tcgagcaaag cccgcttatt ttttacatgc caatacaatg
     5821 taggctgctc tacacctagc ttctgggcga gtttacgggt tgttaaacct tcgattccga
     5881 cctcattaag cagctctaat gcgctgttaa tcactttact tttatctaat ctagacatac
     5941 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6001 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgtttccct cgacaattcg
     6061 cgctagacct agggcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6121 cggttatcca cag
//
