LOCUS       sequence-272562-        4364 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 4364)
  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 4364)
  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: pSiMPla_N + pSiMPla_C
FEATURES             Location/Qualifiers
     source          1..4364
                     /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      2065..2653
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2554..2573
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2807..2824
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2839..2979
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2905..2924)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3065..3087
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3165..3242
                     /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             3243..4325
                     /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(3262..3281)
                     /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 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     1681 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     1741 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     1801 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     1861 ccccagcact cgtccgaggg caaaggaata ataactgtca gaccaagttt actcatatat
     1921 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1981 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2041 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2101 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2161 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     2221 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2281 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2341 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2401 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2461 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2521 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2581 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2641 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2701 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2761 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     2821 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2881 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     2941 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3001 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3061 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3121 cagatcaatt cgcgcgcgaa ggcgaagcgg catgcattta cgttgacacc atcgaatggt
     3181 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     3241 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     3301 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     3361 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     3421 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     3481 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     3541 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     3601 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     3661 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     3721 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     3781 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     3841 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     3901 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     3961 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     4021 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     4081 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     4141 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     4201 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     4261 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     4321 agtgagcgca acgcaattaa tgtaagttag cgcgaattga tctg
//
