LOCUS       sequence-272510-        4562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Maintenance of 2 plasmids with a single antibiotic in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4562)
  AUTHORS   Palanisamy N, Degen A, Morath A, Ballestin Ballestin J, Juraske C, 
            Ozturk MA, Sprenger GA, Youn JW, Schamel WW, Di Ventura B
  TITLE     Split intein-mediated selection of cells containing two plasmids 
            using a single antibiotic.
  JOURNAL   Nat Commun. 2019 Oct 31;10(1):4967. doi: 10.1038/s41467-019-12911-1.
  PUBMED    31672972
REFERENCE   2  (bases 1 to 4562)
  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: "Nat 
            Commun."; date: "2019-10-31"; volume: "10(1):4967. doi"; pages: " 
            10.1038/s41467-019-12911-1"
COMMENT     Sequence Label: pSiMPlk_N + pSiMPlk_C
FEATURES             Location/Qualifiers
     source          1..4562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(193..198,199..215,216..222)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    230..246
                     /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)."
     primer_bind     235..257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             276..983
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTDI
                     ALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAVA
                     KYSNLGGGMDELYK"
     primer_bind     complement(1025..1042)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1195..1281
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1373..1400
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1419..1510
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2263..2851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2752..2771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3005..3022
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3037..3177
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3103..3122)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3263..3285
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3363..3440
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3441..4523
                     /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     complement(3460..3479)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
       61 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
      121 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
      181 tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga
      241 taacaatttc acacaggaaa cagaccatgg aattcgtgtc taagggcgaa gagctgatca
      301 aggaaaatat gcgtatgaag gtggtcatgg aaggttcggt caacggccac caattcaaat
      361 gcacaggtga aggagaaggc agaccgtacg agggagtgca aaccatgagg atcaaagtca
      421 tcgagggagg acccctgcca tttgcctttg acattcttgc cacgtcgttc atgtatggca
      481 gccgtacctt tatcaagtac ccggccgaca tccctgattt ctttaaacag tcctttcctg
      541 agggttttac ttgggaaaga gttacgagat acgaagatgg tggagtcgtc accgtcacgc
      601 aggacaccag ccttgaggat ggcgagctcg tctacaacgt caaggtcaga ggggtaaact
      661 ttccctccaa tggtcccgtg atgcagaaga agaccaaggg ttgggagcct aatacagaga
      721 tgatgtatcc agcagatggt ggtctgagag gatacactga catcgcactg aaagttgatg
      781 gtggtggcca tctgcactgc aacttcgtga caacttacag gtcaaaaaag accgtcggga
      841 acatcaagat gcccggtgtc catgccgttg atcaccgcct ggaaaggatc gaggagagtg
      901 acaatgaaac ctacgtagtg caaagagaag tggcagttgc caaatacagc aaccttggtg
      961 gtggcatgga cgagctgtac aagtaaaagc ttggctgttt tggcggatga gagaagattt
     1021 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
     1081 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
     1141 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
     1201 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     1261 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     1321 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc
     1381 atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca
     1441 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1501 aaggaagagt atgatgctga aaaaaatcct gaaaatcgaa gagctggatg aacgtgaact
     1561 gatcgatatt gaggtgtccg gtaaccacct gttttacgct aacgatattc tgacccacaa
     1621 ctcttcttct aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     1681 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     1741 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     1801 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     1861 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     1921 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     1981 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     2041 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aactgtcaga
     2101 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     2161 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     2221 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2281 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2341 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2401 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2461 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2521 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2581 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2641 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2701 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2761 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2821 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2881 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     2941 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3001 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3061 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     3121 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3181 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3241 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3301 ccgaaacgcg cgaggcagca gatcaattcg cgcgcgaagg cgaagcggca tgcatttacg
     3361 ttgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     3421 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     3481 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     3541 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     3601 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     3661 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     3721 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     3781 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     3841 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     3901 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
     3961 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     4021 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
     4081 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
     4141 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
     4201 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
     4261 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     4321 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     4381 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     4441 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     4501 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagcg cgaattgatc
     4561 tg
//
