LOCUS       sequence-337701-        5345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express RNA-based (p)ppGpp sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5345)
  AUTHORS   Sun Z, Wu R, Zhao B, Zeinert R, Chien P, You M
  TITLE     Live-Cell Imaging of Guanosine Tetra- and Pentaphosphate (p)ppGpp 
            with RNA-based Fluorescent Sensors*.
  JOURNAL   Angew Chem Int Ed Engl. 2021 Nov 2;60(45):24070-24074. doi: 
            10.1002/anie.202111170. Epub 2021 Oct 5.
  PUBMED    34487413
REFERENCE   2  (bases 1 to 5345)
  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: 
            "10.1002/anie.202111170"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2021-11-2- 2"; volume: "60"; issue: "45"; pages: 
            "24070-24074"
COMMENT     Sequence Label: pET-28c S2
FEATURES             Location/Qualifiers
     source          1..5345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..62
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     296..313
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    328..467
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(393..412)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     553..575
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(569..760)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1569..2651)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2613..2632
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2652..2729)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2935..2954
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3038..3057
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3038..3056
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3193..3240
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             3247..3264
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3317..3335)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3331..3378
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3415..3870
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3502..3521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3712..3733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3962..4777)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3962..4777)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4685..4704
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4769..4788
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(4899..5345,1..142)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
       61 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      121 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      181 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      241 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      301 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
      361 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
      421 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
      481 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
      541 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
      601 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
      661 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
      721 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
      781 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
      841 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
      901 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
      961 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     1021 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     1081 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     1141 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     1201 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     1261 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     1321 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     1381 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     1441 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     1501 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     1561 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1621 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     1681 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     1741 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     1801 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     1861 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     1921 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     1981 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     2041 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     2101 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     2161 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     2221 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     2281 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     2341 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     2401 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     2461 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     2521 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     2581 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     2641 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     2701 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     2761 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     2821 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     2881 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     2941 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     3001 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     3061 gtgtacctta gggttccggc cataaggcgt cagcgaccga gcggtacaat gctgtcgagt
     3121 agagtgtggg ctcgcaagag acggtcgggt ccagcaacac cgtgagcata aaaggctcca
     3181 gcggcaagtt ccctagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     3241 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     3301 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     3361 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     3421 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3481 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3541 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3601 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3661 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3721 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3781 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3841 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     3901 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     3961 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     4021 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     4081 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     4141 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     4201 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     4261 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     4321 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     4381 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     4441 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     4501 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     4561 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     4621 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     4681 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     4741 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     4801 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4861 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4921 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4981 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5041 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5101 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5161 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5221 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5281 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5341 gtaag
//
