LOCUS       sequence-324150-        5596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses R. rubrum Form II rubisco and S. elongatus Prk.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5596)
  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 5596)
  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-Rrub-FII-prk
FEATURES             Location/Qualifiers
     source          1..5596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(392..486)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(517..1311)
                     /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(628..647)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1238..1257
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1490..1508
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1529..1547,1548..1553,1554..1570,1571..1576,
                     1577..1602)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1529..1547
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1554..1572
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1612..1623
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3082..3099
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4149..4235
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4408..5031)
                     /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     4939..4958
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(5032..5136)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5312..5596,1..304))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5392..5411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
       61 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      121 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
      181 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      241 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      301 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      361 ttaagggatt ttggtcatga ctagtgcttg gattctcacc aataaaaaac gcccggcggc
      421 aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca
      481 ggagtccaag cgagctctcg aaccccagag tcccgctcag aagaactcgt caagaaggcg
      541 atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc
      601 agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata
      661 gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac
      721 catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat
      781 gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag
      841 atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt
      901 cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc
      961 agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg
     1021 cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc
     1081 gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt
     1141 cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg
     1201 gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct
     1261 ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga
     1321 tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa
     1381 gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg
     1441 caattccgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     1501 cacgaggccc tttcgtcttc acctcgagtc cctatcagtg atagagattg acatccctat
     1561 cagtgataga gatactgagc acatcagcag gacgcactga ccgaattcat taaagaggag
     1621 aaaggtacca tggaccagtc atctcgttac gtcaatctgg cgctcaagga agaggatctg
     1681 atcgccggcg gcgagcatgt gctttgtgcc tatatcatga agcccaaggc cggatatggc
     1741 tatgtggcga ccgcggcgca tttcgccgcc gagagttcga cgggcaccaa cgtcgaggtc
     1801 tgcaccaccg acgatttcac ccggggcgtc gacgccctgg tctatgaggt ggacgaggcc
     1861 cgcgagctga ccaagatcgc ctatccggtg gctttgttcg accgcaacat caccgacggc
     1921 aaggcgatga tcgcctcgtt cctgacgctc accatgggaa acaaccaggg tatgggcgac
     1981 gtggaatacg ccaagatgca cgatttctat gtgcccgagg cttatcgcgc cctgtttgat
     2041 ggcccgagcg tcaatatctc ggccctgtgg aaagtgctgg ggcggcccga ggtcgacggc
     2101 ggtctggtcg tcggcacgat catcaagccg aagctcggcc tgcgtcccaa gcccttcgcc
     2161 gaggcctgcc acgccttctg gctgggcggc gacttcatca agaacgacga gccccagggc
     2221 aatcagccct tcgccccctt gcgcgacacc atcgccctgg tcgccgacgc catgaggcgg
     2281 gcccaggacg agaccggcga ggccaagctg ttctcggcca atatcaccgc cgacgatccc
     2341 ttcgagatca tcgcccgtgg cgagtatgtg ctggagacct tcggcgagaa cgcctcgcat
     2401 gtcgccttgc tggtcgacgg ctatgtcgcc ggcgccgcgg cgatcaccac ggcgcgccgc
     2461 cgcttccccg ataacttctt gcattatcac cgggctggcc acggcgccgt cacctcgccc
     2521 cagtccaagc gcggctatac cgccttcgtc cattgcaaga tggcccgcct tcaaggcgcc
     2581 agcggcatcc acaccggcac catgggcttt ggcaagatgg aaggcgagtc cagcgaccgc
     2641 gccatcgcct atatgctgac ccaggacgag gcccaggggc cgttctaccg tcaatcctgg
     2701 ggcggcatga aggcttgtac gccgatcatc agcggcggca tgaacgccct gcgcatgccc
     2761 ggcttcttcg agaacctggg taatgccaat gtcatcttga ccgccggcgg cggcgccttc
     2821 ggccatatcg acggcccggt ggccggggcg cggtcgttgc gtcaagcctg gcaagcctgg
     2881 cgggacgggg ttccggttct ggactatgcc cgcgagcaca aggaactggc ccgcgccttc
     2941 gagtccttcc ccggcgacgc cgaccagatc tatccgggct ggcgcaaggc cctgggcgtc
     3001 gaggacaccc gcagcgccct tccggcgtaa atctaataga aataattttg tttaacttta
     3061 taagcaggac cggcggcgat gcatcatcac catcaccaca gcaagccaga tcgtgttgtt
     3121 ttgatcggcg ttgccggtga ctccggttgc ggcaaatcaa ccttcctaaa tcgccttgcc
     3181 gacttgtttg gtacggaatt gatgacggtc atctgcttgg atgactatca cagtctcgat
     3241 cgcaagggcc ggaaggaagc aggcgtaacg gctttggatc cccgcgccaa caactttgac
     3301 ttgatgtatg aacaggtcaa ggcgttgaag aacggcgaaa cgatcatgaa gccgatctac
     3361 aaccatgaaa ccggcttgat cgatccgccc gaaaaaatcg aacccaatcg catcattgtg
     3421 atcgagggtc tgcatccgct ttacgacgag cgcgtgcgtg aactgctcga tttcagcgtt
     3481 tacctcgaca tcgatgacga agtcaaaatc gcttggaaga tccaacgcga tatggcagaa
     3541 cgcggccact cctacgaaga tgtcctcgcc tcgatcgaag cgcgccgccc tgacttcaag
     3601 gcctacattg agccccagcg tggccatgcg gacatcgtca tccgcgtcat gccgacccag
     3661 ctaatcccca atgacaccga gcgcaaggtg ctgcgggtgc agttgatcca acgggaaggc
     3721 cgcgatggtt ttgagccggc ttacctgttc gacgaaggtt cgaccatcca gtggacgccc
     3781 tgcggtcgta agctgacctg ctcctatccg ggcattcgct tagcctacgg ccctgacacc
     3841 tactacggtc acgaagtctc agtgcttgag gtcgacggtc agttcgagaa cctcgaagag
     3901 atgatctacg tcgagggcca cctcagcaag accgacacgc agtactacgg tgagttgacc
     3961 cacctgctgc tacagcacaa agattacccg ggttcgaaca acggcacggg tctgttccaa
     4021 gtgctgaccg gcctgaaaat gcgggcggcc tatgagcgtt tgacctccca agcagcaccc
     4081 gtcgccgcta gtgtctagat ggaattcctg cagcccgggg gatcccatgg tacgcgtgct
     4141 agaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     4201 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gccgctagca gttcgtagac
     4261 ggaaacaaac gcagaatcca agcaaaaggg aataagggcg acacggaaat gttgaatact
     4321 catactcttc ctttttcaat attattgaag catttatcag ggttatgtcc actgggttcg
     4381 tgccttcatc cggatatcca ggaactgtta agacccactt tcacatttaa gttgtttttc
     4441 taatccgcat atgatcaatt caaggccgaa taagaaggct ggctctgcac cttggtgatc
     4501 aaataattcg atagcttgtc gtaataatgg cggcatacta tcagtagtag gtgtttccct
     4561 ttcttcttta gcgacttgat gctcttgatc ttccaatacg caacctaaag taaaatgccc
     4621 cacagcgctg agtgcatata atgcattctc tagtgaaaaa ccttgttggc ataaaaaggc
     4681 taattgattt tcgagagttt catactgttt ttctgtaggc cgtgtaccta aatgtacttt
     4741 tgctccatcg cgatgactta gtaaagcaca tctaaaactt ttagcgttat tacgtaaaaa
     4801 atcttgccag ctttcccctt ctaaagggca aaagtgagta tggtgcctat ctaacatctc
     4861 aatggctaag gcgtcgagca aagcccgctt attttttaca tgccaataca atgtaggctg
     4921 ctctacacct agcttctggg cgagtttacg ggttgttaaa ccttcgattc cgacctcatt
     4981 aagcagctct aatgcgctgt taatcacttt acttttatct aatctagaca tactcttcct
     5041 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5101 atgtatttag aaaaataaac aaataggggt tccgcgtttc cctcgacaat tcgcgctaga
     5161 cctagggcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5221 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5281 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5341 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5401 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5461 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5521 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5581 ttcagcccga ccgctg
//
