LOCUS       sequence-324170-        6060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses R. spaeroides Form IC rubisco and S. elongatus Prk.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6060)
  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 6060)
  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-Rs-FI-prk
FEATURES             Location/Qualifiers
     source          1..6060
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     440..459
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     692..710
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(731..749,750..755,756..772,773..778,779..804)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    731..749
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    756..774
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             814..825
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2748..2765
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3815..3901
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4074..4697)
                     /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     4605..4624
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(4698..4802)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4978..5566)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5058..5077)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5654..5748)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(5779..6060,1..513))
                     /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(5890..5909)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
       61 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
      121 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
      181 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag
      241 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
      301 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc
      361 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
      421 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
      481 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg
      541 atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac
      601 tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg acgtctaaga
      661 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
      721 tcacctcgag tccctatcag tgatagagat tgacatccct atcagtgata gagatactga
      781 gcacatcagc aggacgcact gaccgaattc attaaagagg agaaaggtac catggatacc
      841 aagaccaccg agatcaaggg caaggaacgc tacaaggccg gcgtcctgaa atatgcccag
      901 atgggttact gggacggcga ttacgtcccc aaggacaccg acgtgctggc gctgttccgc
      961 atcacaccgc aggagggggt ggatccggtc gaggcggccg ccgccgtggc cggcgaaagc
     1021 tcgactgcga cctggaccgt ggtctggacc gaccggctga ccgcctgcga cagctaccgc
     1081 gccaaggcct accgggtgga gccggtgccg ggcacgccgg ggcagtattt ctgctatgtg
     1141 gcctatgacc tgatcctgtt cgaggaaggc tcgatcgcca acctcaccgc ctcgatcatc
     1201 ggcaacgtct tcagcttcaa gccgctgaag gccgcccggc tcgaggacat gcgcttcccc
     1261 gtggcctatg tgaagaccta caagggcccg cccaccggca tcgtgggcga gcgcgagcgg
     1321 ctggacaagt tcggcaagcc ccttctcggc gccaccacga agcccaagct cggcctgtcg
     1381 ggcaagaact acggccgcgt ggtctatgag gggctgaagg gcggcctcga cttcatgaag
     1441 gacgacgaga acatcaactc gcagcccttc atgcactggc gcgaccggtt cctctatgtg
     1501 atggaggcgg tgaacctcgc ctcggcccag accggcgagg tgaagggcca ctacctcaac
     1561 atcaccgccg gcacgatgga ggagatgtac cggcgggcgg aattcgccaa gtcgcttggc
     1621 tcggtcatcg tgatggtcga tctcatcatc ggctatacgg cgatccagtc gatctcggaa
     1681 tggtgccggc agaacgacat gatcctgcac atgcaccgcg cgggccacgg cacctacacg
     1741 cggcagaaga accacggcat cagcttccgc gtgatcgcca agtggctgcg gctggcgggg
     1801 gtggaccatc tgcattgcgg cacggccgtg ggcaagctcg agggcgaccc gctgacggtg
     1861 cagggctatt acaatgtctg ccgcgagccc ttcaacacgg tcgacctgcc gcgcgggatc
     1921 ttcttcgagc aggactgggc ggacctgagg aaggtcatgc cggtggcctc gggcgggatc
     1981 catgccgggc agatgcacca gcttctgagc ctcttcggcg acgatgtggt gctgcagttc
     2041 ggcggcggca ccatcggcca cccgatgggg atccaggccg gggcgaccgc gaaccgcgtg
     2101 gcgctggaag cgatggtgct ggcgcgcaac gagggccgca acatcgatgt ggagggcccg
     2161 gagatcctcc gcgccgcggc caaatggtgc aagccgctgg aggccgccct cgatacctgg
     2221 ggcaacatca ccttcaacta cacctccacc gacacgtcgg acttcgtgcc gaccgcctcg
     2281 gtcgcgatgt aaggagaccg gacatgcgca tcacccaagg ctgcttctcg ttcctgcccg
     2341 acctgaccga cgcgcagatc tcggcgcagg tggactattg cctcggccgc ggctgggccg
     2401 tgagcctcga acataccgac gacccgcatc cccggaacac ctactgggaa atgtggggaa
     2461 tgccgatgtt cgacctgcgc gaccccaagg gcgtgatgat cgagctggac gagtgccgca
     2521 aggcctggcc cggccgctac atccgcatca atgccttcga ttccacccgc ggcttcgaga
     2581 cggtcacgat gagcttcatc gtcaaccgcc ccgaggtcga gcccagcctc cgcatggagc
     2641 gcaccgaggt cgacggccgg tcgatccgct acacgcacag catcgtccgc tgataaatct
     2701 aatagaaata attttgttta actttataag caggaccggc ggcgatgcat catcaccatc
     2761 accacagcaa gccagatcgt gttgttttga tcggcgttgc cggtgactcc ggttgcggca
     2821 aatcaacctt cctaaatcgc cttgccgact tgtttggtac ggaattgatg acggtcatct
     2881 gcttggatga ctatcacagt ctcgatcgca agggccggaa ggaagcaggc gtaacggctt
     2941 tggatccccg cgccaacaac tttgacttga tgtatgaaca ggtcaaggcg ttgaagaacg
     3001 gcgaaacgat catgaagccg atctacaacc atgaaaccgg cttgatcgat ccgcccgaaa
     3061 aaatcgaacc caatcgcatc attgtgatcg agggtctgca tccgctttac gacgagcgcg
     3121 tgcgtgaact gctcgatttc agcgtttacc tcgacatcga tgacgaagtc aaaatcgctt
     3181 ggaagatcca acgcgatatg gcagaacgcg gccactccta cgaagatgtc ctcgcctcga
     3241 tcgaagcgcg ccgccctgac ttcaaggcct acattgagcc ccagcgtggc catgcggaca
     3301 tcgtcatccg cgtcatgccg acccagctaa tccccaatga caccgagcgc aaggtgctgc
     3361 gggtgcagtt gatccaacgg gaaggccgcg atggttttga gccggcttac ctgttcgacg
     3421 aaggttcgac catccagtgg acgccctgcg gtcgtaagct gacctgctcc tatccgggca
     3481 ttcgcttagc ctacggccct gacacctact acggtcacga agtctcagtg cttgaggtcg
     3541 acggtcagtt cgagaacctc gaagagatga tctacgtcga gggccacctc agcaagaccg
     3601 acacgcagta ctacggtgag ttgacccacc tgctgctaca gcacaaagat tacccgggtt
     3661 cgaacaacgg cacgggtctg ttccaagtgc tgaccggcct gaaaatgcgg gcggcctatg
     3721 agcgtttgac ctcccaagca gcacccgtcg ccgctagtgt ctagatggaa ttcctgcagc
     3781 ccgggggatc ccatggtacg cgtgctagag gcatcaaata aaacgaaagg ctcagtcgaa
     3841 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     3901 tccgccgccg ctagcagttc gtagacggaa acaaacgcag aatccaagca aaagggaata
     3961 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4021 tatcagggtt atgtccactg ggttcgtgcc ttcatccgga tatccaggaa ctgttaagac
     4081 ccactttcac atttaagttg tttttctaat ccgcatatga tcaattcaag gccgaataag
     4141 aaggctggct ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa taatggcggc
     4201 atactatcag tagtaggtgt ttccctttct tctttagcga cttgatgctc ttgatcttcc
     4261 aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg catataatgc attctctagt
     4321 gaaaaacctt gttggcataa aaaggctaat tgattttcga gagtttcata ctgtttttct
     4381 gtaggccgtg tacctaaatg tacttttgct ccatcgcgat gacttagtaa agcacatcta
     4441 aaacttttag cgttattacg taaaaaatct tgccagcttt ccccttctaa agggcaaaag
     4501 tgagtatggt gcctatctaa catctcaatg gctaaggcgt cgagcaaagc ccgcttattt
     4561 tttacatgcc aatacaatgt aggctgctct acacctagct tctgggcgag tttacgggtt
     4621 gttaaacctt cgattccgac ctcattaagc agctctaatg cgctgttaat cactttactt
     4681 ttatctaatc tagacatact cttccttttt caatattatt gaagcattta tcagggttat
     4741 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4801 cgtttccctc gacaattcgc gctagaccta gggcgttcgg ctgcggcgag cggtatcagc
     4861 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4921 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4981 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5041 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5101 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5161 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5221 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5281 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5341 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5401 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5461 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5521 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5581 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5641 gactagtgct tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca
     5701 aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca agcgagctct
     5761 cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     5821 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag
     5881 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag
     5941 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca
     6001 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc
//
