LOCUS       sequence-378525-        4467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of protein N tagged with mKate from a T7-lac inducible 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4467)
  AUTHORS   Guo H, Ryan JC, Song X, Mallet A, Zhang M, Pabst V, Decrulle AL, 
            Ejsmont P, Wintermute EH, Lindner AB
  TITLE     Spatial engineering of E. coli with addressable phase-separated 
            RNAs.
  JOURNAL   Cell. 2022 Sep 29;185(20):3823-3837.e23. doi: 
            10.1016/j.cell.2022.09.016.
  PUBMED    36179672
REFERENCE   2  (bases 1 to 4467)
  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.cell.2022.09"; journalName: "Cell"; date: "2022-09-29- 
            29"; volume: "185"; issue: "20"; pages: "3823-3837.e23"
COMMENT     Sequence Label: pPN-mKate (PT7LacO-PN-mKate)
FEATURES             Location/Qualifiers
     source          1..4467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2..67
                     /codon_start=1
                     /product="N-terminal RNA-binding domain from the lambda 
                     bacteriophage antiterminator protein N (Baron-Benhamou et 
                     al., 2004)"
                     /label=lambda N peptide
                     /translation="MDAQTRRRERRAEKQAQWKAAN"
     CDS             341..1036
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     primer_bind     complement(1065..1083)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1079..1126
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1297..2088)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2089..2180)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2228..2966)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(3176..4258)
                     /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     4220..4239
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4259..4336)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4382..4401
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4382..4400
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4401..4425
                     /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)."
ORIGIN
        1 catggatgca caaacacgcc gtcgtgaacg tcgtgcggag aagcaggccc aatggaaggc
       61 agcaaatcct cttcttgtgg gggtgagtgc caaacctgtt aatcgtccta ttctttcgtt
      121 aaatcgcaag cctaagtctc gcgtggagtc cgcattgaac ccgattgatc ttactgtatt
      181 ggcggagtat cacaagcaga tcgaatctaa cctgcaacgt attgagcgca agaatcagcg
      241 cacctggtac tctaagccag gcgaacgcgg aatcacttgc agtggccgcc agaaaatcaa
      301 agggaagtca atcccactta ttggcggatc aggaggtggc atgtctgagc tgattaagga
      361 gaacatgcac atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac
      421 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggcggtcga
      481 gggcggccct ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa
      541 aaccttcatc aaccacaccc agggcatccc cgacttcttt aagcagtcct tccctgaggg
      601 cttcacatgg gagagagtca ccacatacga agatgggggc gtgctgaccg ctacccagga
      661 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc
      721 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagacgct
      781 gtaccccgct gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg
      841 gggccacctg atctgcaact taaagaccac atacagatcc aagaaacccg ctaagaacct
      901 caagatgccc ggcgtctact atgtggacag acgactggaa agaatcaagg aggccgacaa
      961 agaaacctac gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa
     1021 actggggcac agatgataag aattcttaac ctaggctgct gccaccgctg agcaataact
     1081 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc
     1141 atttgagaag cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata
     1201 gacataagcg gctatttaac gaccctgccc tgaaccgacg accgggtcat cgtggccgga
     1261 tcttgcggcc cctcggcttg aacgaattgt tagacattat ttgccgacta ccttggtgat
     1321 ctcgcctttc acgtagtgga caaattcttc caactgatct gcgcgcgagg ccaagcgatc
     1381 ttcttcttgt ccaagataag cctgtctagc ttcaagtatg acgggctgat actgggccgg
     1441 caggcgctcc attgcccagt cggcagcgac atccttcggc gcgattttgc cggttactgc
     1501 gctgtaccaa atgcgggaca acgtaagcac tacatttcgc tcatcgccag cccagtcggg
     1561 cggcgagttc catagcgtta aggtttcatt tagcgcctca aatagatcct gttcaggaac
     1621 cggatcaaag agttcctccg ccgctggacc taccaaggca acgctatgtt ctcttgcttt
     1681 tgtcagcaag atagccagat caatgtcgat cgtggctggc tcgaagatac ctgcaagaat
     1741 gtcattgcgc tgccattctc caaattgcag ttcgcgctta gctggataac gccacggaat
     1801 gatgtcgtcg tgcacaacaa tggtgacttc tacagcgcgg agaatctcgc tctctccagg
     1861 ggaagccgaa gtttccaaaa ggtcgttgat caaagctcgc cgcgttgttt catcaagcct
     1921 tacggtcacc gtaaccagca aatcaatatc actgtgtggc ttcaggccgc catccactgc
     1981 ggagccgtac aaatgtacgg ccagcaacgt cggttcgaga tggcgctcga tgacgccaac
     2041 tacctctgat agttgagtcg atacttcggc gatcaccgct tccctcatac tcttcctttt
     2101 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2161 tatttagaaa aataaacaaa tagctagctc actcggtcgc tacgctccgg gcgtgagact
     2221 gcggcgggcg ctgcggacac atacaaagtt acccacagat tccgtggata agcaggggac
     2281 taacatgtga ggcaaaacag cagggccgcg ccggtggcgt ttttccatag gctccgccct
     2341 cctgccagag ttcacataaa cagacgcttt tccggtgcat ctgtgggagc cgtgaggctc
     2401 aaccatgaat ctgacagtac gggcgaaacc cgacaggact taaagatccc caccgtttcc
     2461 ggcgggtcgc tccctcttgc gctctcctgt tccgaccctg ccgtttaccg gatacctgtt
     2521 ccgcctttct cccttacggg aagtgtggcg ctttctcata gctcacacac tggtatctcg
     2581 gctcggtgta ggtcgttcgc tccaagctgg gctgtaagca agaactcccc gttcagcccg
     2641 actgctgcgc cttatccggt aactgttcac ttgagtccaa cccggaaaag cacggtaaaa
     2701 cgccactggc agcagccatt ggtaactggg agttcgcaga ggatttgttt agctaaacac
     2761 gcggttgctc ttgaagtgtg cgccaaagtc cggctacact ggaaggacag atttggttgc
     2821 tgtgctctgc gaaagccagt taccacggtt aagcagttcc ccaactgact taaccttcga
     2881 tcaaaccacc tccccaggtg gttttttcgt ttacagggca aaagattacg cgcagaaaaa
     2941 aaggatctca agaagatcct ttgatctttt ctactgaacc gctctagatt tcagtgcaat
     3001 ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg taattctcat
     3061 gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     3121 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     3181 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3241 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     3301 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     3361 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     3421 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     3481 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     3541 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     3601 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     3661 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     3721 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     3781 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     3841 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     3901 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     3961 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     4021 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     4081 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     4141 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     4201 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     4261 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     4321 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaaa
     4381 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctgt agaaataatt
     4441 ttgtttaact ttaataagga gatatac
//
