LOCUS       sequence-198909-        8168 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. Kan resistance 
            marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8168)
  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 8168)
  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-3
FEATURES             Location/Qualifiers
     source          1..8168
                     /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(4533..5327)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4644..4663)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5254..5273
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5420..5491)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(5603..6316)
                     /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             6405..6413
                     /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    6428..6446
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     rep_origin      6673..7444
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             7445..8107
                     /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 gatccgatga
     4501 agttttgacg gtatcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag
     4561 aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc
     4621 cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg
     4681 tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg
     4741 atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatccgc
     4801 gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca
     4861 tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct
     4921 tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc
     4981 atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact
     5041 tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa
     5101 ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcttggag ttcattcagg
     5161 gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac
     5221 acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc
     5281 acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct
     5341 catcctgtct cttgatccac tagattattg aagcatttat cagggttatt gtctcatgag
     5401 cggatacata ttcggtacca ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5461 attgtctcat gagcggatac atatttgaat gaattcaacc atttactatg ttatgttctg
     5521 aggggagtga aaattcccct aattcgatga agattcttgc tcaattgtta tcagctatgc
     5581 gccgaccaga acaccttgcc gatcagccaa acgtctcttc aggccactga ctagcgataa
     5641 ctttccccac aacggaacaa ctctcattgc atgggatcat tgggtactgt gggtttagtg
     5701 gttgtaaaaa cacctgaccg ctatccctga tcagtttctt gaaggtaaac tcatcacccc
     5761 caagtctggc tatgcagaaa tcacctggct caacagcctg ctcagggtca acgagaatta
     5821 acattccgtc aggaaagctt ggcttggagc ctgttggtgc ggtcatggaa ttaccttcaa
     5881 cctcaagcca gaatgcagaa tcactggctt ttttggttgt gcttacccat ctctccgcat
     5941 cacctttggt aaaggttcta agctcaggtg agaacatccc tgcctgaaca tgagaaaaaa
     6001 cagggtactc atactcactt ctaagtgacg gctgcatact aaccgcttca tacatctcgt
     6061 agatttctct ggcgattgaa gggctaaatt cttcaacgct aactttgaga atttttgtaa
     6121 gcaatgcggc gttataagca tttaatgcat tgatgccatt aaataaagca ccaacgcctg
     6181 actgccccat ccccatcttg tctgcgacag attcctggga taagccaagt tcatttttct
     6241 ttttttcata aattgcttta aggcgacgtg cgtcctcaag ctgctcttgt gttaatggtt
     6301 tcttttttgt gctcatacgt taaatctatc accgcaaggg ataaatatct aacaccgtgc
     6361 gtgttgacta ttttacctct ggcggtgata atggttgcat gtactaagga ggttgtatgg
     6421 aacaacgaga tgtgtataag agacagctgg cctgcccctc ccttttggtg tccaaccggc
     6481 tcgacggggg cagcgcaagg cggtgcctcc ggcgggccac tcaatgcttg agtatactca
     6541 ctagactttg cttcgcaaag tcgtgaccgc ctacggcggc tgcggcgccc tacgggcttg
     6601 ctctccgggc ttcgccctgc gcggtcgctg cgctcccttg ccagcccgtg gatatgtgga
     6661 cgatggccgc gagcggccac cggctggctc gcttcgctcg gcccgtggac aaccctgctg
     6721 gacaagctga tggacaggct gcgcctgccc acgagcttga ccacagggat tgcccaccgg
     6781 ctacccagcc ttcgaccaca tacccaccgg ctccaactgc gcggcctgcg gccttgcccc
     6841 atcaattttt ttaattttct ctggggaaaa gcctccggcc tgcggcctgc gcgcttcgct
     6901 tgccggttgg acaccaagtg gaaggcgggt caaggctcgc gcagcgaccg cgcagcggct
     6961 tggccttgac gcgcctggaa cgacccaagc ctatgcgagt gggggcagtc gaagggcgaa
     7021 gcccgcccgc ctgccccccg agcctcacgg cggcgagtgc gggggttcca agggggcagc
     7081 gccaccttgg gcaaggccga aggccgcgca gtcgatcaac aagccccgga ggggccactt
     7141 tttgccggag ggggagccgc gccgaaggcg tgggggaacc ccgcaggggt gcccttcttt
     7201 gggcaccaaa gaactagata tagggcgaaa tgcgaaagac ttaaaaatca acaacttaaa
     7261 aaaggggggt acgcaacagc tcattgcggc accccccgca atagctcatt gcgtaggtta
     7321 aagaaaatct gtaattgact gccactttta cgcaacgcat aattgttgtc gcgctgccga
     7381 aaagttgcag ctgattgcgc atggtgccgc aaccgtgcgg cacccctacc gcatggagat
     7441 aagcatggcc acgcagtcca gagaaatcgg cattcaagcc aagaacaagc ccggtcactg
     7501 ggtgcaaacg gaacgcaaag cgcatgaggc gtgggccggg cttattgcga ggaaacccac
     7561 ggcggcaatg ctgctgcatc acctcgtggc gcagatgggc caccagaacg ccgtggtggt
     7621 cagccagaag acactttcca agctcatcgg acgttctttg cggacggtcc aatacgcagt
     7681 caaggacttg gtggccgagc gctggatctc cgtcgtgaag ctcaacggcc ccggcaccgt
     7741 gtcggcctac gtggtcaatg accgcgtggc gtggggccag ccccgcgacc agttgcgcct
     7801 gtcggtgttc agtgccgccg tggtggttga tcacgacgac caggacgaat cgctgttggg
     7861 gcatggcgac ctgcgccgca tcccgaccct gtatccgggc gagcagcaac taccgaccgg
     7921 ccccggcgag gagccgccca gccagcccgg cattccgggc atggaaccag acctgccagc
     7981 cttgaccgaa acggaggaat gggaacggcg cgggcagcag cgcctgccga tgcccgatga
     8041 gccgtgtttt ctggacgatg gcgagccgtt ggagccgccg acacgggtca cgctgccgcg
     8101 ccggtagcac ttgggttgcg cagcaacccg taagtgcgct gttccagact atcggctgta
     8161 ccgcctcg
//
