LOCUS       sequence-259619-        6426 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 6426)
  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 6426)
  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: pSiMPlh_N + pSiMPlh_C
FEATURES             Location/Qualifiers
     source          1..6426
                     /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      3024..3479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3111..3130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        complement(5078..5180)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5572..5589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5706..6251)
                     /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(5786..5805)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6323..6342
                     /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 gttggggcaa actattaact
     2701 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     2761 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     2821 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     2881 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     2941 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3001 tcatatatac tttagattga tttacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3061 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3121 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3181 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     3241 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3301 ggagtccacg ttctttaata gtggactctt gttccaaact tgaacaacac tcaaccctat
     3361 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3421 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaattta
     3481 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     3541 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3601 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3661 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3721 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3781 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtcaggcat ttgagaagca
     3841 cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga cataagcggc
     3901 tatttaacga ccctgccctg aaccgacgac cgggtcgaat ttgctttcga atttctgcca
     3961 ttcatccgct tattatcact tattcaggcg tagcaccagg cgtttaaggg caccaataac
     4021 tgccttaaaa aaattattct ttaacataca gacacatacc ttctttcaga ccaccggaga
     4081 tgttcatttc accagtctgc gtcggaaaca ggtgttcttc ggaacagatg atttccttgc
     4141 catcttccag ggtgattttg taagactttt ttttagattt cgggaagacg ttcagaactt
     4201 cgttgtaacc cgtgttgctc agtaccagat caccgacctg gatgttggaa atttccttca
     4261 taccctgcgg ggtctgaacc tgggttttca gatccaagca gtaaccagac tcctgatgcc
     4321 tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc cagaagaaga
     4381 tgttggcgac ctcgtattgg gaatccccga acatcgcctc gctccagtca atgaccgctg
     4441 ttatgcggcc attgtccgtc aggacattgt tggagccgaa atccgcgtgc acgaggtgcc
     4501 ggacttcggg gcagtcctcg gcccaaagca tcagctcatc gagagcctgc gcgacggacg
     4561 cactgacggt gtcgtccatc acagtttgcc agtgatacac atggggatca gcaatcgcgc
     4621 atatgaaatc acgccatgta gtgtattgac cgattccttg cggtccgaat gggccgaacc
     4681 cgctcgtctg gctaagatcg gccgcagcga tcgcatccat agcctccgcg accggttgta
     4741 gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt gaacggcggg
     4801 agatgcaata ggtcaggctc tcgctaaact ccccaatgtc aagcacttcc ggaatcggga
     4861 gcgcggccga tgcaaagtgc cgataaacat aacgatcttt gtagaaacca tcggcgcagc
     4921 tatttacccg caggacatat ccacgccctc ctacatcgaa gctgaaagca cgagattctt
     4981 cgccctccga gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc agaaacttct
     5041 cgacagacgt cgcggtgagt tcaggctttt tacccatttt agcttcctta gctcctgaaa
     5101 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
     5161 aacctcttac gtgccgatca acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg
     5221 tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc acaggtattt
     5281 attcggcgca aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt atgatggtgt
     5341 ttttgaggtg ctccagtggc ttctgtttct atcagctgtc cctcctgttc agctactgac
     5401 ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctagcggag tgtatactgg
     5461 cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa
     5521 ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt cctcgctcac
     5581 tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg
     5641 agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag
     5701 ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag
     5761 tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc
     5821 gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt atggccgcgt
     5881 ttgtctcatt ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg
     5941 tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta tccggtaact atcgtcttga
     6001 gtccaacccg gaaagacatg caaaagcacc actggcagca gccactggta attgatttag
     6061 aggagttagt cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca agttttggtg
     6121 actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag agaaccttcg
     6181 aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa
     6241 aacgatctca agaagatcat cttattaatc agataaaata tttgctcatg agcccgaagt
     6301 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     6361 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctgctca tgtttgacag
     6421 cttatc
//
