LOCUS       sequence-324151-        5986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses H. neapolitanus CbbLS and S. elongatus Prk. Rescues CCMB1 
            in M9 glycerol at 10% CO2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5986)
  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 5986)
  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: p1A
FEATURES             Location/Qualifiers
     source          1..5986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     266..285
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     518..536
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(557..575,576..581,582..598,599..604,605..630)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    557..575
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    582..600
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             640..651
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2500..2517
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3567..3653
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3826..4449)
                     /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     4357..4376
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(4450..4554)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4730..5318)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4810..4829)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5406..5500)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(5531..5986,1..339))
                     /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(5642..5661)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
       61 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
      121 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
      181 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
      241 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
      301 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
      361 ctcttgatca gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca
      421 gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccgacgt
      481 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
      541 tcgtcttcac ctcgagtccc tatcagtgat agagattgac atccctatca gtgatagaga
      601 tactgagcac atcagcagga cgcactgacc gaattcatta aagaggagaa aggtaccatg
      661 gcagttaaaa agtatagtgc tggtgtaaaa gaataccggc agacctattg gatgccggaa
      721 tacacaccgt tggattccga catccttgca tgcttcaaaa tcaccccaca accgggtgtt
      781 gatcgcgaag aagccgcagc cgcggttgca gcagaatctt caaccggcac atggaccacc
      841 gtgtggaccg atttgctgac cgacatggac tactacaaag gccgtgccta tcgcattgaa
      901 gacgtacccg gtgacgatgc ggcattctat gcctttatcg cctacccaat cgacctgttc
      961 gaagaagggt cagttgttaa cgtgtttacc tcactggttg gtaacgtgtt cggcttcaaa
     1021 gcggtacgcg gcctgcgtct ggaagatgtt cgcttcccac tcgcctacgt taaaacctgt
     1081 ggcggcccac cgcacggtat tcaagtcgaa cgcgacaaga tgaacaaata tggtcgccca
     1141 ctgttgggtt gcaccatcaa gccaaaactt ggtttgtctg cgaaaaacta cggccgtgcc
     1201 gtatacgagt gcctccgtgg cggcctcgac ttcactaaag atgatgaaaa catcaattct
     1261 cagccgttca tgcgctggcg cgatcgcttc ttgttcgtac aagacgcgac cgaaactgct
     1321 gaagcccaaa ccggcgaacg caaaggccat tacctcaacg taacggcgcc aactcctgaa
     1381 gaaatgtaca agcgcgcaga attcgccaaa gaaattggcg cgccaatcat tatgcacgac
     1441 tacatcaccg gtggcttcac ggccaacact ggcttggcca agtggtgtca agacaacggc
     1501 gtactgctgc acatccaccg tgcgatgcat gcggttatcg accgtaaccc gaaccacggt
     1561 attcacttcc gtgttctgac caagattctg cgtttgtcgg gtggcgatca cctgcacacc
     1621 ggtaccgttg tcggcaaact ggaaggcgac cgtgcctcta ctctgggctg gattgatttg
     1681 ctccgcgaat cgtttatccc tgaagatcgc tcgcgcggta tcttcttcga tcaagactgg
     1741 ggttcaatgc caggcgtatt cgctgtggcc tctggtggta ttcacgtatg gcacatgcct
     1801 gcgctggtta acatctttgg tgacgactct gtcctccaat tcggtggcgg tacgctgggt
     1861 catccatggg gcaacgctgc cggtgctgct gccaaccgtg ttgctctgga agcctgcgta
     1921 gaagcgcgta accaaggccg cgatatcgaa aaagaaggca aagaaattct gactgctgct
     1981 gcacagcaca gcccagaact gaagattgcc atggaaactt ggaaagagat caaattcgaa
     2041 tttgacactg tcgacaaact cgacactcaa aatcgttgat ccctcgtacc acacaacata
     2101 ctaaggtgag taaccatggc tgaaatgcag gattacaagc aaagcctcaa atatgagact
     2161 ttctcttatc ttccacccat gaacgcggaa cgcatccgcg ctcaaatcaa gtacgcaatt
     2221 gctcaaggct ggagccccgg cattgagcac gtagaagtga aaaactccat gaaccaatat
     2281 tggtacatgt ggaaacttcc cttcttcggc gaacaaaatg tcgacaacgt gttggctgaa
     2341 attgaagcgt gtcgtagtgc gtatccaaca caccaggtca aactggtggc ttatgacaac
     2401 tatgcgcaaa gcttaggtct ggccttcgtg gtctaccgcg gcaactaaat ctaatagaaa
     2461 taattttgtt taactttata agcaggaccg gcggcgatgc atcatcacca tcaccacagc
     2521 aagccagatc gtgttgtttt gatcggcgtt gccggtgact ccggttgcgg caaatcaacc
     2581 ttcctaaatc gccttgccga cttgtttggt acggaattga tgacggtcat ctgcttggat
     2641 gactatcaca gtctcgatcg caagggccgg aaggaagcag gcgtaacggc tttggatccc
     2701 cgcgccaaca actttgactt gatgtatgaa caggtcaagg cgttgaagaa cggcgaaacg
     2761 atcatgaagc cgatctacaa ccatgaaacc ggcttgatcg atccgcccga aaaaatcgaa
     2821 cccaatcgca tcattgtgat cgagggtctg catccgcttt acgacgagcg cgtgcgtgaa
     2881 ctgctcgatt tcagcgttta cctcgacatc gatgacgaag tcaaaatcgc ttggaagatc
     2941 caacgcgata tggcagaacg cggccactcc tacgaagatg tcctcgcctc gatcgaagcg
     3001 cgccgccctg acttcaaggc ctacattgag ccccagcgtg gccatgcgga catcgtcatc
     3061 cgcgtcatgc cgacccagct aatccccaat gacaccgagc gcaaggtgct gcgggtgcag
     3121 ttgatccaac gggaaggccg cgatggtttt gagccggctt acctgttcga cgaaggttcg
     3181 accatccagt ggacgccctg cggtcgtaag ctgacctgct cctatccggg cattcgctta
     3241 gcctacggcc ctgacaccta ctacggtcac gaagtctcag tgcttgaggt cgacggtcag
     3301 ttcgagaacc tcgaagagat gatctacgtc gagggccacc tcagcaagac cgacacgcag
     3361 tactacggtg agttgaccca cctgctgcta cagcacaaag attacccggg ttcgaacaac
     3421 ggcacgggtc tgttccaagt gctgaccggc ctgaaaatgc gggcggccta tgagcgtttg
     3481 acctcccaag cagcacccgt cgccgctagt gtctagatgg aattcctgca gcccggggga
     3541 tcccatggta cgcgtgctag aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     3601 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgc
     3661 cgctagcagt tcgtagacgg aaacaaacgc agaatccaag caaaagggaa taagggcgac
     3721 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     3781 ttatgtccac tgggttcgtg ccttcatccg gatatccagg aactgttaag acccactttc
     3841 acatttaagt tgtttttcta atccgcatat gatcaattca aggccgaata agaaggctgg
     3901 ctctgcacct tggtgatcaa ataattcgat agcttgtcgt aataatggcg gcatactatc
     3961 agtagtaggt gtttcccttt cttctttagc gacttgatgc tcttgatctt ccaatacgca
     4021 acctaaagta aaatgcccca cagcgctgag tgcatataat gcattctcta gtgaaaaacc
     4081 ttgttggcat aaaaaggcta attgattttc gagagtttca tactgttttt ctgtaggccg
     4141 tgtacctaaa tgtacttttg ctccatcgcg atgacttagt aaagcacatc taaaactttt
     4201 agcgttatta cgtaaaaaat cttgccagct ttccccttct aaagggcaaa agtgagtatg
     4261 gtgcctatct aacatctcaa tggctaaggc gtcgagcaaa gcccgcttat tttttacatg
     4321 ccaatacaat gtaggctgct ctacacctag cttctgggcg agtttacggg ttgttaaacc
     4381 ttcgattccg acctcattaa gcagctctaa tgcgctgtta atcactttac ttttatctaa
     4441 tctagacata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4501 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgtttccc
     4561 tcgacaattc gcgctagacc tagggcgttc ggctgcggcg agcggtatca gctcactcaa
     4621 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4681 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4741 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4801 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4861 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4921 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4981 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5041 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5101 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5161 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5221 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5281 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5341 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgactagtg
     5401 cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa caaatccaga
     5461 tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct ctcgaacccc
     5521 agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg
     5581 gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag
     5641 caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac
     5701 agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc
     5761 catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt
     5821 cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt
     5881 ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag
     5941 ccggatcaag cgtatgcagc cgccgcattg catcagccat gatgga
//
