LOCUS       sequence-324169-        5986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  p1A negative control expressing inactive CbbLS (carbamylated K194M 
            mutation) and Prk.
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 CbbL-
FEATURES             Location/Qualifiers
     source          1..5986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(163..751)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(243..262)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(839..933)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(964..1758)
                     /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(1075..1094)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1685..1704
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1937..1955
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1976..1994,1995..2000,2001..2017,2018..2023,
                     2024..2049)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1976..1994
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2001..2019
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2059..2070
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3919..3936
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4986..5072
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5245..5868)
                     /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     5776..5795
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(5869..5973)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttcgcgctag acctagggcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
       61 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
      121 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
      181 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      241 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      301 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      361 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      421 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      481 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
      541 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
      601 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
      661 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
      721 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
      781 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgacta gtgcttggat
      841 tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc agatggagtt
      901 ctgaggtcat tactggatct atcaacagga gtccaagcga gctctcgaac cccagagtcc
      961 cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc
     1021 gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc
     1081 acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat
     1141 gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt
     1201 cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg
     1261 cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg
     1321 agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc
     1381 aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag
     1441 gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc
     1501 ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag
     1561 ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag
     1621 aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg
     1681 ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa
     1741 tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag atcttgatcc
     1801 cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc
     1861 aaccttacca gagggcgccc cagctggcaa ttccgacgtc taagaaacca ttattatcat
     1921 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcacc tcgagtccct
     1981 atcagtgata gagattgaca tccctatcag tgatagagat actgagcaca tcagcaggac
     2041 gcactgaccg aattcattaa agaggagaaa ggtaccatgg cagttaaaaa gtatagtgct
     2101 ggtgtaaaag aataccggca gacctattgg atgccggaat acacaccgtt ggattccgac
     2161 atccttgcat gcttcaaaat caccccacaa ccgggtgttg atcgcgaaga agccgcagcc
     2221 gcggttgcag cagaatcttc aaccggcaca tggaccaccg tgtggaccga tttgctgacc
     2281 gacatggact actacaaagg ccgtgcctat cgcattgaag acgtacccgg tgacgatgcg
     2341 gcattctatg cctttatcgc ctacccaatc gacctgttcg aagaagggtc agttgttaac
     2401 gtgtttacct cactggttgg taacgtgttc ggcttcaaag cggtacgcgg cctgcgtctg
     2461 gaagatgttc gcttcccact cgcctacgtt aaaacctgtg gcggcccacc gcacggtatt
     2521 caagtcgaac gcgacaagat gaacaaatat ggtcgcccac tgttgggttg caccatcaag
     2581 ccaaaacttg gtttgtctgc gaaaaactac ggccgtgccg tatacgagtg cctccgtggc
     2641 ggcctcgact tcactatgga tgatgaaaac atcaattctc agccgttcat gcgctggcgc
     2701 gatcgcttct tgttcgtaca agacgcgacc gaaactgctg aagcccaaac cggcgaacgc
     2761 aaaggccatt acctcaacgt aacggcgcca actcctgaag aaatgtacaa gcgcgcagaa
     2821 ttcgccaaag aaattggcgc gccaatcatt atgcacgact acatcaccgg tggcttcacg
     2881 gccaacactg gcttggccaa gtggtgtcaa gacaacggcg tactgctgca catccaccgt
     2941 gcgatgcatg cggttatcga ccgtaacccg aaccacggta ttcacttccg tgttctgacc
     3001 aagattctgc gtttgtcggg tggcgatcac ctgcacaccg gtaccgttgt cggcaaactg
     3061 gaaggcgacc gtgcctctac tctgggctgg attgatttgc tccgcgaatc gtttatccct
     3121 gaagatcgct cgcgcggtat cttcttcgat caagactggg gttcaatgcc aggcgtattc
     3181 gctgtggcct ctggtggtat tcacgtatgg cacatgcctg cgctggttaa catctttggt
     3241 gacgactctg tcctccaatt cggtggcggt acgctgggtc atccatgggg caacgctgcc
     3301 ggtgctgctg ccaaccgtgt tgctctggaa gcctgcgtag aagcgcgtaa ccaaggccgc
     3361 gatatcgaaa aagaaggcaa agaaattctg actgctgctg cacagcacag cccagaactg
     3421 aagattgcca tggaaacttg gaaagagatc aaattcgaat ttgacactgt cgacaaactc
     3481 gacactcaaa atcgttgatc cctcgtacca cacaacatac taaggtgagt aaccatggct
     3541 gaaatgcagg attacaagca aagcctcaaa tatgagactt tctcttatct tccacccatg
     3601 aacgcggaac gcatccgcgc tcaaatcaag tacgcaattg ctcaaggctg gagccccggc
     3661 attgagcacg tagaagtgaa aaactccatg aaccaatatt ggtacatgtg gaaacttccc
     3721 ttcttcggcg aacaaaatgt cgacaacgtg ttggctgaaa ttgaagcgtg tcgtagtgcg
     3781 tatccaacac accaggtcaa actggtggct tatgacaact atgcgcaaag cttaggtctg
     3841 gccttcgtgg tctaccgcgg caactaaatc taatagaaat aattttgttt aactttataa
     3901 gcaggaccgg cggcgatgca tcatcaccat caccacagca agccagatcg tgttgttttg
     3961 atcggcgttg ccggtgactc cggttgcggc aaatcaacct tcctaaatcg ccttgccgac
     4021 ttgtttggta cggaattgat gacggtcatc tgcttggatg actatcacag tctcgatcgc
     4081 aagggccgga aggaagcagg cgtaacggct ttggatcccc gcgccaacaa ctttgacttg
     4141 atgtatgaac aggtcaaggc gttgaagaac ggcgaaacga tcatgaagcc gatctacaac
     4201 catgaaaccg gcttgatcga tccgcccgaa aaaatcgaac ccaatcgcat cattgtgatc
     4261 gagggtctgc atccgcttta cgacgagcgc gtgcgtgaac tgctcgattt cagcgtttac
     4321 ctcgacatcg atgacgaagt caaaatcgct tggaagatcc aacgcgatat ggcagaacgc
     4381 ggccactcct acgaagatgt cctcgcctcg atcgaagcgc gccgccctga cttcaaggcc
     4441 tacattgagc cccagcgtgg ccatgcggac atcgtcatcc gcgtcatgcc gacccagcta
     4501 atccccaatg acaccgagcg caaggtgctg cgggtgcagt tgatccaacg ggaaggccgc
     4561 gatggttttg agccggctta cctgttcgac gaaggttcga ccatccagtg gacgccctgc
     4621 ggtcgtaagc tgacctgctc ctatccgggc attcgcttag cctacggccc tgacacctac
     4681 tacggtcacg aagtctcagt gcttgaggtc gacggtcagt tcgagaacct cgaagagatg
     4741 atctacgtcg agggccacct cagcaagacc gacacgcagt actacggtga gttgacccac
     4801 ctgctgctac agcacaaaga ttacccgggt tcgaacaacg gcacgggtct gttccaagtg
     4861 ctgaccggcc tgaaaatgcg ggcggcctat gagcgtttga cctcccaagc agcacccgtc
     4921 gccgctagtg tctagatgga attcctgcag cccgggggat cccatggtac gcgtgctaga
     4981 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     5041 tgtcggtgaa cgctctcctg agtaggacaa atccgccgcc gctagcagtt cgtagacgga
     5101 aacaaacgca gaatccaagc aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5161 actcttcctt tttcaatatt attgaagcat ttatcagggt tatgtccact gggttcgtgc
     5221 cttcatccgg atatccagga actgttaaga cccactttca catttaagtt gtttttctaa
     5281 tccgcatatg atcaattcaa ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa
     5341 taattcgata gcttgtcgta ataatggcgg catactatca gtagtaggtg tttccctttc
     5401 ttctttagcg acttgatgct cttgatcttc caatacgcaa cctaaagtaa aatgccccac
     5461 agcgctgagt gcatataatg cattctctag tgaaaaacct tgttggcata aaaaggctaa
     5521 ttgattttcg agagtttcat actgtttttc tgtaggccgt gtacctaaat gtacttttgc
     5581 tccatcgcga tgacttagta aagcacatct aaaactttta gcgttattac gtaaaaaatc
     5641 ttgccagctt tccccttcta aagggcaaaa gtgagtatgg tgcctatcta acatctcaat
     5701 ggctaaggcg tcgagcaaag cccgcttatt ttttacatgc caatacaatg taggctgctc
     5761 tacacctagc ttctgggcga gtttacgggt tgttaaacct tcgattccga cctcattaag
     5821 cagctctaat gcgctgttaa tcactttact tttatctaat ctagacatac tcttcctttt
     5881 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5941 tatttagaaa aataaacaaa taggggttcc gcgtttccct cgacaa
//
