LOCUS       sequence-348362-        6269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet inducible, KAN Resistant, fluor labelled (YFP) synthesis domain 
            of the E. coli relA gene on a low copy backbone. Used to induce 
            controllable synthesis of ppGpp in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6269)
  AUTHORS   Buke F, Grilli J, Cosentino Lagomarsino M, Bokinsky G, Tans SJ
  TITLE     ppGpp is a bacterial cell size regulator.
  JOURNAL   Curr Biol. 2022 Feb 28;32(4):870-877.e5. doi: 
            10.1016/j.cub.2021.12.033. Epub 2022 Jan 5.
  PUBMED    34990598
REFERENCE   2  (bases 1 to 6269)
  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"; doi: 
            "10.1016/j.cub.2021.12.033"; journalName: "Curr Biol"; date: 
            "2022-02-28- 28"; volume: "32"; issue: "4"; pages: "870-877.e5"
COMMENT     Sequence Label: pRelA*
FEATURES             Location/Qualifiers
     source          1..6269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(125..144)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     472..493
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      571..642
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      658..685
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1216..2166)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     terminator      complement(2928..3022)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3053..3847)
                     /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(3164..3183)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3774..3793
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3990..4616)
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     4521..4540
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        4632..4687
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    4668..4686
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(6088..6090,6091..6093,6094..6269,1..538)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6133..6154)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 cgtgaccacc ctgggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca
       61 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
      121 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
      181 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
      241 gctggagtac aactacaaca gccacaacgt ctatatcacc gccgacaagc agaagaacgg
      301 catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga
      361 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
      421 cctgagctac cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
      481 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaagg
      541 atcctaactc gagtaaggat ctccaggcat caaataaaac gaaaggctca gtcgaaagac
      601 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc
      661 tcaccttcgg gtgggccttt ctgcgtttat acctagggta cgggttttgc tgcccgcaaa
      721 cgggctgttc tggtgttgct agtttgttat cagaatcgca gatccggctt cagccggttt
      781 gccggctgaa agcgctattt cttccagaat tgccatgatt ttttccccac gggaggcgtc
      841 actggctccc gtgttgtcgg cagctttgat tcgataagca gcatcgcctg tttcaggctg
      901 tctatgtgtg actgttgagc tgtaacaagt tgtctcaggt gttcaatttc atgttctagt
      961 tgctttgttt tactggtttc acctgttcta ttaggtgtta catgctgttc atctgttaca
     1021 ttgtcgatct gttcatggtg aacagctttg aatgcaccaa aaactcgtaa aagctctgat
     1081 gtatctatct tttttacacc gttttcatct gtgcatatgg acagttttcc ctttgatatg
     1141 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca
     1201 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg
     1261 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact
     1321 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt
     1381 ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc
     1441 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc
     1501 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt
     1561 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa
     1621 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat
     1681 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat
     1741 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa
     1801 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa
     1861 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc
     1921 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct
     1981 actgatgttc atcatctgag cgtattggtt ataagtgaac gataccgtcc gttctttcct
     2041 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc
     2101 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc
     2161 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag
     2221 tcaatgataa ttactagtcc ttttcccggg tgatctgggt atctgtaaat tctgctagac
     2281 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg
     2341 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa
     2401 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta
     2461 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa
     2521 aggcttaagt agcaccctcg caagctcggg caaatcgctg aatattcctt ttgtctccga
     2581 ccatcaggca cctgagtcgc tgtctttttc gtgacattca gttcgctgcg ctcacggctc
     2641 tggcagtgaa tgggggtaaa tggcactaca ggcgcctttt atggattcat gcaaggaaac
     2701 tacccataat acaagaaaag cccgtcacgg gcttctcagg gcgttttatg gcgggtctgc
     2761 tatgtggtgc tatctgactt tttgctgttc agcagttcct gccctctgat tttccagtct
     2821 gaccacttcg gattatcccg tgacaggtca ttcagactgg ctaatgcacc cagtaaggca
     2881 gcggtatcat caacaggctt acccgtctta ctgtccctag tgcttggatt ctcaccaata
     2941 aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt
     3001 actggatcta tcaacaggag tccaagcgag ctctcgaacc ccagagtccc gctcagaaga
     3061 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
     3121 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
     3181 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
     3241 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
     3301 cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct
     3361 gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
     3421 gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
     3481 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
     3541 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
     3601 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
     3661 cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
     3721 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
     3781 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
     3841 caatcatgcg aaacgatcct catcctgtct cttgatcaga tcatgatccc ctgcgccatc
     3901 agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag
     3961 agggcgcccc agctggcaat tccgacgtct taagacccac tttcacattt aagttgtttt
     4021 tctaatccgc atatgatcaa ttcaaggccg aataagaagg ctggctctgc accttggtga
     4081 tcaaataatt cgatagcttg tcgtaataat ggcggcatac tatcagtagt aggtgtttcc
     4141 ctttcttctt tagcgacttg atgctcttga tcttccaata cgcaacctaa agtaaaatgc
     4201 cccacagcgc tgagtgcata taatgcattc tctagtgaaa aaccttgttg gcataaaaag
     4261 gctaattgat tttcgagagt ttcatactgt ttttctgtag gccgtgtacc taaatgtact
     4321 tttgctccat cgcgatgact tagtaaagca catctaaaac ttttagcgtt attacgtaaa
     4381 aaatcttgcc agctttcccc ttctaaaggg caaaagtgag tatggtgcct atctaacatc
     4441 tcaatggcta aggcgtcgag caaagcccgc ttatttttta catgccaata caatgtaggc
     4501 tgctctacac ctagcttctg ggcgagttta cgggttgtta aaccttcgat tccgacctca
     4561 ttaagcagct ctaatgcgct gttaatcact ttacttttat ctaatctaga catcattaat
     4621 tcctaatttt tgttgacact ctatcgttga tagagttatt ttaccactcc ctatcagtga
     4681 tagagaaaag aattcaaaag atctaggagg aaaaaaatgg ttgcggtaag aagtgcacat
     4741 atcaataagg ctggtgaatt tgatccggaa aaatggatcg caagtctggg tattaccagc
     4801 cagaagtcgt gtgagtgctt agccgaaacc tgggcgtatt gtctgcaaca gacgcagggg
     4861 catccggatg ccagtctgtt attgtggcgt ggtgttgaga tggtggagat cctctcgaca
     4921 ttaagtatgg acattgacac gctgcgggcg gcgctgcttt tccctctggc ggatgccaac
     4981 gtagtcagcg aagatgtgct gcgtgagagc gtcggtaagt cggtcgttaa ccttattcac
     5041 ggcgtgcgtg atatggcggc gatccgccag ctgaaagcga cgcacactga ttctgtttcc
     5101 tccgaacagg tcgataacgt tcgccggatg ttattggcga tggtcgatga ttttcgctgc
     5161 gtagtcatca aactggcgga gcgtattgct catctgcgcg aagtaaaaga tgcgccggaa
     5221 gatgaacgtg tactggcggc aaaagagtgt accaacatct acgcaccgct ggctaaccgt
     5281 ctcggaatcg gacaactgaa atgggaactg gaagattact gcttccgtta cctccatcca
     5341 accgaataca aacgaattgc caaactgctg catgaacggc gtctcgaccg cgaacactac
     5401 atcgaagagt tcgttggtca tctgcgcgct gagatgaaag ctgaaggcgt taaagcggaa
     5461 gtgtatggtc gtccgaaaca catctacagc atctggcgta aaatgcagaa aaagaacctc
     5521 gcctttgatg agctgtttga tgtgcgtgcg gtacgtattg tcgccgagcg tttacaggat
     5581 tgctatgccg cactggggat agtgcacact cactatcgcc acctgccgga tgagtttgac
     5641 gattacgtcg ctaacccgaa accaaacggt tatcagtcta ttcataccgt ggttctgggg
     5701 ccgggtggaa aaaccgttga gatccaaatc cgcaccaaac agatgcatga agatgcagag
     5761 ttgggtgttg ctgcgcactg gaaatataaa gagggcgcgg ctgctggcgg cgcacgttcg
     5821 ggacatgaag accggattgc ctggctgcgt aaactgattg cgtggcagga agagatggct
     5881 gattccggcg aaatgctcga cgaagtacgt agtcaggtct ttgacgaccg ggtgtacgtc
     5941 tttacgccga aaggtgatgt cgttgatttg cctgcgggat caacgccgct ggacttcgct
     6001 taccacatcc acagtgatgt cggacaccgc tgcatcgggg caaaaattgg cgggcgcatt
     6061 gtgccgttca cctaccagct gggatctatg gtgagcaagg gcgaggagct gttcaccggg
     6121 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     6181 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagctgat ctgcaccacc
     6241 ggcaagctgc ccgtgccctg gcccaccct
//
