LOCUS       sequence-198908-        8116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Lambda Red recombinases and a dominant negative MutL 
            allele all controlled by temperature sensitve cI857 repressor for 
            high precision and efficiency MAGE experiments. Cam resistance 
            marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8116)
  AUTHORS   Nyerges A, Csorgo B, Nagy I, Balint B, Bihari P, Lazar V, Apjok G, 
            Umenhoffer K, Bogos B, Posfai G, Pal C
  TITLE     A highly precise and portable genome engineering method allows 
            comparison of mutational effects across bacterial species.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Feb 16. pii: 201520040.
  PUBMED    26884157
REFERENCE   2  (bases 1 to 8116)
  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: "Proc Natl 
            Acad Sci U S A. 2016 Feb 16. pii: 201520040."
COMMENT     Sequence Label: pORTMAGE-4
FEATURES             Location/Qualifiers
     source          1..8116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    4..22
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             complement(2014..2694)
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGEDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     CDS             complement(2691..3476)
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     protein_bind    3909..3923
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     CDS             complement(4523..5182)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPKFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5097..5116
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5183..5285)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(5368..5439)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(5551..6264)
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     RBS             6353..6361
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     misc_feature    6376..6394
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     rep_origin      6621..7392
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             7393..8055
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
ORIGIN
        1 cagctgtctc ttatacacat ctccgctgtg ctttcagtgg atttcggata acagaaaggc
       61 cgggaaatac ccagcctcgc tttgtaacgg agtagacgaa agtgattgcg cctacccgga
      121 tattatcgtg aggatgctca tcgccatttc actcatcttt cagggctttt atcgccggat
      181 gtaaatcaac agattgtaac agaccacccg gcggcgtttt cacaagttgc ggacataacc
      241 gttccacgtc cgccagcagg gttatggcct gtgccattga ccactgcgca tgttcgctca
      301 tcagatttcg tgcaatccac tgcgcaatat tgccaggttc gaatacggac tgcttcgcca
      361 ggtagcctat cagttcagga atcaagattt gtaaattttg ttggcgtaag ggtaaaggca
      421 ctgccctgat ggtcacatgc tgtgcatctg actggaaatc aatacccaat tccgccaggg
      481 cagactgcgc tttttctaat gccgattttt cttcggcaga aacttttagc cgcaacggaa
      541 tcagcagcgg ctgggcgcaa acgggcgctt cacccggcgt caattgtgcc tgacgcagcc
      601 aacgttctgc cactggcaag gataaaagtg aaatgttgcc gtcgcgctcc agcaacgcac
      661 agtcggaatg gacgatagtc agtacccgac caaaactctg actgttcgcc gcaagtgcag
      721 gttcctgcgg ttccggcgct tttaattttt gcatcggcgc gggcgtttgc aaaagctggc
      781 gatacacttc accttgctgt ttctggtagc ctggctgcgc attcggccag ggggcagccg
      841 gacgactgcc tgatgctggc gcaggagtgt agcgcggagc taccggctca cgagctgccg
      901 gttctgcaaa gtgattgcgc cccgccgcca cgcggttttc cggaatggaa cgcggtgcag
      961 gttggggttc atcgtccagc ggtagcggcg tttccagttg ctgttgtagc acgctcagca
     1021 cgccctgata gataaaatca tgcaccagac gcgactgatg gaaacgcact tcgtgtttgg
     1081 cggggtgcac gttgacgtcc acctgatgtg ggtcgatctc cagatacaac acaaatgccg
     1141 gttgctgatc ggcccccagt ttgtcttcgc aggcctggcg gatcgcgtga ttgatcaggc
     1201 gatcgcgcat catgcgaccg ttcacgtagc aatactgaat ttctgccagt gcgggcgtgg
     1261 tgtgatttgg atcggccacc cagccgcgta gcgtgagatc gccgtgttgc cattcaatcg
     1321 ccagcgcttg ttcaagaaaa gcggtgccgc aaatcgcgcc taagcgccgt tctttttgcc
     1381 cgccttccgg cactgcgcgg tactgacgca caattttacc gttatgcgac aggttgatcg
     1441 tgacgtcgaa acgcgccagc gcaatgcggc ggatgatctc atcaatgtgg ttaaattcgg
     1501 ttttctcggt gcgcaggaat ttgcgccgcg ccggggtgtt gtagaacaga tccagcacct
     1561 ccagcgtcgt ccccacagga tgcgccgccg gttttaccgt cacgttcata tcgcgccctt
     1621 cggcataggc ctgccaggct tcctgctgtt ctgcggtgcg tgaagtgagc gtcaggcggg
     1681 aaaccgaact gatactcgcc agcgcctcac cgcgaaagcc caggctgata atggcttcga
     1741 gatcgtccag agaggcgatt ttactggtgg catgacgagc cagcgccagc gccagctcat
     1801 cttttttgat accgcagccg ttatcacgaa tgcggataag tttcgcccca ccgcgttcga
     1861 tatcaatatc gatacgcgtc gcacctgcat cgaggctgtt tttcactagt tctttgacta
     1921 ccgacgcagg tcgctcgacc acctcacctg cggcaatctg gttcgccagt tgtggcggta
     1981 agacctgaat tggcattttg taatcctcct ctctcatcgc cattgctccc caaatacaaa
     2041 accaatttca gccagtgcct cgtccatttt ttcgatgaac tccggcacga tctcgtcaaa
     2101 actcgccatg tacttttcat cccgctcaat cacgacataa tgcaggcctt cacgcttcat
     2161 acgcgggtca tagttggcaa agtaccaggc attttttcgc gtcacccaca tgctgtactg
     2221 cacctgggcc atgtaagctg actttatggc ctcgaaacca ccgagccgga acttcatgaa
     2281 atcccgggag gtaaacgggc atttcagttc aaggccgttg ccgtcactgc ataaaccatc
     2341 gggagagcag gcggtacgca tactttcgtc gcgatagatg atcggggatt cagtaacatt
     2401 cacgccggaa gtaaattcaa acagggttct ggcgtcgttc tcgtactgtt ttccccaggc
     2461 cagtgcttta gcgttaactt ccggagccac accggtgcaa acctcagcaa gcagggtgtg
     2521 gaagtaggac attttcatgt caggccactt ctttccggag cggggttttg ctatcacgtt
     2581 gtgaacttct gaagcggtga tgacgccgag ccgtaatttg tgccacgcat cttccccctg
     2641 ttcgacagct ctcacatcga tcccggtacg ctgcaggata atgtccggtg tcatgctgcc
     2701 accttctgct ctgcggcttt ctgtttcagg aatccaagag cttttactgc ttcggcctgt
     2761 gtcagttctg acgatgcacg aatgtcgcgg cgaaatatct gggaacagag cggcaataag
     2821 tcgtcatccc atgttttatc cagggcgatc agcagagtgt taatctcctg catggtttca
     2881 tcgttaaccg gagtgatgtc gcgttccggc tgacgttctg cagtgtatgc agtattttcg
     2941 acaatgcgct cggcttcatc cttgtcatag ataccagcaa atccgaaggc cagacgggca
     3001 cactgaatca tggctttatg acgtaacatc cgtttgggat gcgactgcca cggccccgtg
     3061 atttctctgc cttcgcgagt tttgaatggt tcgcggcggc attcatccat ccattcggta
     3121 acgcagatcg gatgattacg gtccttgcgg taaatccggc atgtacagga ttcattgtcc
     3181 tgctcaaagt ccatgccatc aaactgctgg ttttcattga tgatgcggga ccagccatca
     3241 acgcccacca ccggaacgat gccattctgc ttatcaggaa aggcgtaaat ttctttcgtc
     3301 cacggattaa ggccgtactg gttggcaacg atcagtaatg cgatgaactg cgcatcgctg
     3361 gcatcacctt taaatgccgt ctggcgaaga gtggtgatca gttcctgtgg gtcgacagaa
     3421 tccatgccga cacgttcagc cagcttccca gccagcgttg cgagtgcagt actcattcgt
     3481 tttatacctc tgaatcaata tcaacctggt ggtgagcaat ggtttcaacc atgtaccgga
     3541 tgtgttctgc catgcgctcc tgaaactcaa catcgtcatc aaacgcacgg gtaatggatt
     3601 ttttgctggc cccgtggcgt tgcaaatgat cgatgcatag cgattcaaac aggtgctggg
     3661 gcaggccttt ttccatgtcg tctgccagtt ctgcctcttt ctcttcacgg gcgagctgct
     3721 ggtagtgacg cgcccagctc tgagcctcaa gacgatcctg aatgtaataa gcgttcatgg
     3781 ctgaactcct gaaatagctg tgaaaatatc gcccgcgaaa tgccgggctg attagtaatc
     3841 cggaatcgca cttacggcca atgcttcgtt tcgtatcaca caccccaaag ccttctgctt
     3901 tgaatgctgc ccttcttcag ggcttaattt ttaagagcgt caccttcatg gtggtcagtg
     3961 cgtcctgctg atgtgctcag tatcaccgcc agtggtattt atgtcaacac cgccagagat
     4021 aatttatcac cgcagatggt tatctgtatg ttttttatat gaatttattt tttgcagggg
     4081 ggcattgttt ggtaggtgag agatctgaat tgctatgttt agtgagttgt atctatttat
     4141 ttttcaataa atacaattgg ttatgtgttt tgggggcgat cgtgaggcaa agaaaacccg
     4201 gcgctgaggc cgggttaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4261 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4321 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4381 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4441 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttag gatccgaggc
     4501 accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta
     4561 attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg
     4621 ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg
     4681 cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat
     4741 tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac
     4801 cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt
     4861 cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa
     4921 cactatccca tatcaccagc tcaccgtctt tcattgccat acggaatttc ggatgagcat
     4981 tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta
     5041 cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa
     5101 ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat
     5161 atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa
     5221 aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc
     5281 gatcaacgtc tcattttcgc caaaagttgg cccagggctt cccggtatca acagggacac
     5341 caggatttat ttattctgcg cggtaccact cttccttttt caatattatt gaagcattta
     5401 tcagggttat tgtctcatga gcggatacat atttgaatga attcaaccat ttactatgtt
     5461 atgttctgag gggagtgaaa attcccctaa ttcgatgaag attcttgctc aattgttatc
     5521 agctatgcgc cgaccagaac accttgccga tcagccaaac gtctcttcag gccactgact
     5581 agcgataact ttccccacaa cggaacaact ctcattgcat gggatcattg ggtactgtgg
     5641 gtttagtggt tgtaaaaaca cctgaccgct atccctgatc agtttcttga aggtaaactc
     5701 atcaccccca agtctggcta tgcagaaatc acctggctca acagcctgct cagggtcaac
     5761 gagaattaac attccgtcag gaaagcttgg cttggagcct gttggtgcgg tcatggaatt
     5821 accttcaacc tcaagccaga atgcagaatc actggctttt ttggttgtgc ttacccatct
     5881 ctccgcatca cctttggtaa aggttctaag ctcaggtgag aacatccctg cctgaacatg
     5941 agaaaaaaca gggtactcat actcacttct aagtgacggc tgcatactaa ccgcttcata
     6001 catctcgtag atttctctgg cgattgaagg gctaaattct tcaacgctaa ctttgagaat
     6061 ttttgtaagc aatgcggcgt tataagcatt taatgcattg atgccattaa ataaagcacc
     6121 aacgcctgac tgccccatcc ccatcttgtc tgcgacagat tcctgggata agccaagttc
     6181 atttttcttt ttttcataaa ttgctttaag gcgacgtgcg tcctcaagct gctcttgtgt
     6241 taatggtttc ttttttgtgc tcatacgtta aatctatcac cgcaagggat aaatatctaa
     6301 caccgtgcgt gttgactatt ttacctctgg cggtgataat ggttgcatgt actaaggagg
     6361 ttgtatggaa caacgagatg tgtataagag acagctggcc tgcccctccc ttttggtgtc
     6421 caaccggctc gacgggggca gcgcaaggcg gtgcctccgg cgggccactc aatgcttgag
     6481 tatactcact agactttgct tcgcaaagtc gtgaccgcct acggcggctg cggcgcccta
     6541 cgggcttgct ctccgggctt cgccctgcgc ggtcgctgcg ctcccttgcc agcccgtgga
     6601 tatgtggacg atggccgcga gcggccaccg gctggctcgc ttcgctcggc ccgtggacaa
     6661 ccctgctgga caagctgatg gacaggctgc gcctgcccac gagcttgacc acagggattg
     6721 cccaccggct acccagcctt cgaccacata cccaccggct ccaactgcgc ggcctgcggc
     6781 cttgccccat caattttttt aattttctct ggggaaaagc ctccggcctg cggcctgcgc
     6841 gcttcgcttg ccggttggac accaagtgga aggcgggtca aggctcgcgc agcgaccgcg
     6901 cagcggcttg gccttgacgc gcctggaacg acccaagcct atgcgagtgg gggcagtcga
     6961 agggcgaagc ccgcccgcct gccccccgag cctcacggcg gcgagtgcgg gggttccaag
     7021 ggggcagcgc caccttgggc aaggccgaag gccgcgcagt cgatcaacaa gccccggagg
     7081 ggccactttt tgccggaggg ggagccgcgc cgaaggcgtg ggggaacccc gcaggggtgc
     7141 ccttctttgg gcaccaaaga actagatata gggcgaaatg cgaaagactt aaaaatcaac
     7201 aacttaaaaa aggggggtac gcaacagctc attgcggcac cccccgcaat agctcattgc
     7261 gtaggttaaa gaaaatctgt aattgactgc cacttttacg caacgcataa ttgttgtcgc
     7321 gctgccgaaa agttgcagct gattgcgcat ggtgccgcaa ccgtgcggca cccctaccgc
     7381 atggagataa gcatggccac gcagtccaga gaaatcggca ttcaagccaa gaacaagccc
     7441 ggtcactggg tgcaaacgga acgcaaagcg catgaggcgt gggccgggct tattgcgagg
     7501 aaacccacgg cggcaatgct gctgcatcac ctcgtggcgc agatgggcca ccagaacgcc
     7561 gtggtggtca gccagaagac actttccaag ctcatcggac gttctttgcg gacggtccaa
     7621 tacgcagtca aggacttggt ggccgagcgc tggatctccg tcgtgaagct caacggcccc
     7681 ggcaccgtgt cggcctacgt ggtcaatgac cgcgtggcgt ggggccagcc ccgcgaccag
     7741 ttgcgcctgt cggtgttcag tgccgccgtg gtggttgatc acgacgacca ggacgaatcg
     7801 ctgttggggc atggcgacct gcgccgcatc ccgaccctgt atccgggcga gcagcaacta
     7861 ccgaccggcc ccggcgagga gccgcccagc cagcccggca ttccgggcat ggaaccagac
     7921 ctgccagcct tgaccgaaac ggaggaatgg gaacggcgcg ggcagcagcg cctgccgatg
     7981 cccgatgagc cgtgttttct ggacgatggc gagccgttgg agccgccgac acgggtcacg
     8041 ctgccgcgcc ggtagcactt gggttgcgca gcaacccgta agtgcgctgt tccagactat
     8101 cggctgtacc gcctcg
//
