LOCUS       sequence-343182-        6104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express RNA-based (p)ppGpp sensor and a NirFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6104)
  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 6104)
  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: pETDuet S2
FEATURES             Location/Qualifiers
     source          1..6104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1350..1368
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      1589..1635
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1660..1678
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1679..1703
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     CDS             1746..2450
                     /codon_start=1
                     /product="near-infrared fluorescent protein eqFP670"
                     /label=NirFP
                     /note="mammalian codon-optimized"
                     /translation="MGEDSELISENMHTKLYMEGTVNGHHFKCTSEGEGKPYEGTQTCK
                     IKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGGV
                     LTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEANTEMLYPADSGLRGHNQM
                     ALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRKLERIKEADKETYVEQHEMAVA
                     RYCDLPSKLGHS"
     CDS             2463..2507
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2545..2563)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2559..2606
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2643..3098
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2730..2749)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2940..2961
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3213..4004,4005..4073))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3836..3855
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4074..4166)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4247..4835
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4736..4755
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4989..5006
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5021..5160
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5086..5105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5246..5268
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5262..5453)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6025..6104,1..1003))
                     /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
                     EAYKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttataggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgcgatc ccgcgaaatt aatacgactc actatagggg aagtgtacct
     1381 tagggttccg gccataaggc gtcagcgacc gagcggtaca atgctgtcga gtagagtgtg
     1441 ggctcgcaag agacggtcgg gtccagcaac accgtgagca taaaaggctc cagcggcaag
     1501 ttccctagca taaccccttg gggcctctaa acgggtcttg aggggttttt gccgcgggtc
     1561 cagggttcaa gtccctgttc gggcgccata gcataacccc ttggggcctc taaacgggtc
     1621 ttgaggggtt ttttgcgtac acagccgcat aatcgaaatt aatacgactc actatagggg
     1681 aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata
     1741 tacatatggg agaggatagc gagctgatct ccgagaacat gcacacgaaa ctgtacatgg
     1801 agggcaccgt gaacggccac cacttcaagt gcacatccga gggcgaaggc aagccctacg
     1861 agggcaccca gacctgtaag atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
     1921 acatcctggc taccagcttc atgtacggca gcaaaacctt tatcaaccac acccagggca
     1981 tccccgactt ctttaagcag tccttccctg agggcttcac atgggagagg atcaccacat
     2041 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccagaac ggctgcctca
     2101 tctacaacgt caagatcaac ggggtgaact tcccatccaa cggccctgtg atgcagaaga
     2161 aaacactcgg ctgggaggcc aacaccgaga tgctgtaccc cgctgacagc ggtctgagag
     2221 gccataatca gatggccctg aagctcgtgg gcgggggcta cctgcactgc tccctcaaga
     2281 ccacatacag atccaagaaa cccgctaaga acctcaagat gcccggcttc tacttcgtgg
     2341 accgtaaact ggaaagaatc aaggaggccg acaaagagac ctacgtcgag cagcacgaga
     2401 tggctgtggc caggtactgc gacctgccta gcaaactggg gcacagctga ctcgagtctg
     2461 gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag
     2521 cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2581 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     2641 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2701 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2761 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2821 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2881 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2941 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3001 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3061 taacgcgaat tttaacaaaa tattaacgtt tacaatttct ggcggcacga tggcatgaga
     3121 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3181 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3241 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3301 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3361 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3421 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3481 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3541 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3601 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3661 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3721 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3781 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3841 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3901 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3961 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4021 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4081 ctttttcaat catgattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4141 tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa aatcccttaa cgtgagtttt
     4201 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4261 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4321 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4381 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4441 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4501 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4561 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4621 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4681 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4741 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4801 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4861 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4921 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4981 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     5041 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     5101 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     5161 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5221 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5281 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     5341 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     5401 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     5461 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     5521 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     5581 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     5641 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     5701 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     5761 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     5821 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     5881 cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca ccggaaggag
     5941 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     6001 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6061 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gttt
//
