LOCUS       sequence-272493-        6021 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 6021)
  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 6021)
  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..6021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(96..974)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1001..1285
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1208..1227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             join(1327..1329,1330..1332,1333..2046)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1327..1329,1330..1332,1333..2043)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1372..1393)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1633..1652)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1980..2001
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2085..2102)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2255..2341
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2433..2460
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2479..2570
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3028..3483
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3115..3134)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3325..3346
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4576..4595
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4669..4771)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5163..5180)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5297..5842)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(5377..5396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5918..5937
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac
       61 tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca
      121 ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta
      181 aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata
      241 ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag
      301 cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag
      361 caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg
      421 tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct
      481 tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc
      541 ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc
      601 gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca
      661 tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga
      721 tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa
      781 acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata
      841 taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc
      901 ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt
      961 tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat
     1021 tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta
     1081 accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt
     1141 aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca
     1201 ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta
     1261 tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcgaatt cgagctcaag
     1321 gccaggatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1381 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1441 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1501 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1561 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1621 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1681 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1741 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1801 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1861 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1921 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1981 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2041 aagtaaaagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
     2101 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     2161 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     2221 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     2281 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     2341 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     2401 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
     2461 tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2521 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2581 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2641 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca gcaaactatt
     2701 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2761 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2821 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2881 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2941 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3001 ttactcatat atactttaga ttgatttacg cgccctgtag cggcgcatta agcgcggcgg
     3061 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3121 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3181 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3241 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3301 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3361 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3421 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3481 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3541 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3601 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3661 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3721 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3781 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtcag gcatttgaga
     3841 agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag
     3901 cggctattta acgaccctgc cctgaaccga cgaccgggtc gaatttgctt tcgaatttct
     3961 gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt aagggcacca
     4021 ataactgcct taaaaaaatt attctttaac atacagacac ataccttctt tcagaccacc
     4081 ggagatgttc atttcaccag tctgcgtcgg aaacaggtgt tcttcggaac agatgatttc
     4141 cttgccatct tccagggtga ttttgtaaga ctttttttta gatttcggga agacgttcag
     4201 aacttcgttg taacccgtgt tgctcagtac cagatcaccg acctggatgt tggaaatttc
     4261 cttcataccc tgcggggtct gaacctgggt tttcagatcc aagcagtaac cagattcacc
     4321 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     4381 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     4441 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     4501 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     4561 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     4621 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcattt tagcttcctt
     4681 agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg
     4741 gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggccca
     4801 gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt
     4861 cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg
     4921 tatgatggtg tttttgaggt gctccagtgg cttctgtttc tatcagctgt ccctcctgtt
     4981 cagctactga cggggtggtg cgtaacggca aaagcaccgc cggacatcag cgctagcgga
     5041 gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc
     5101 aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct
     5161 tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac
     5221 gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg
     5281 ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac
     5341 gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5401 gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt
     5461 tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc
     5521 aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac
     5581 tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt
     5641 aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac
     5701 aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca
     5761 gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac
     5821 gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat atttctaggc
     5881 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5941 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatct
     6001 gctcatgttt gacagcttat c
//
