LOCUS       sequence-457485-        8096 bp    DNA     circular SYN 14-APR-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. Ap resistance 
            marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8096)
  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 8096)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-2
FEATURES             Location/Qualifiers
     source          1..8096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      619..1390
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     misc_feature    1617..1635
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     RBS             1650..1658
                     /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)"
     CDS             1747..2460
                     /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"
     promoter        2572..2643
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2644..2712,2713..3504)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2862..2881)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    4068..4082
                     /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             4515..5300
                     /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"
     CDS             5297..5977
                     /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"
     misc_feature    7969..7987
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             complement(join(8052..8096,1..618))
                     /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 gccatcgtcc agaaaacacg gctcatcggg catcggcagg cgctgctgcc cgcgccgttc
       61 ccattcctcc gtttcggtca aggctggcag gtctggttcc atgcccggaa tgccgggctg
      121 gctgggcggc tcctcgccgg ggccggtcgg tagttgctgc tcgcccggat acagggtcgg
      181 gatgcggcgc aggtcgccat gccccaacag cgattcgtcc tggtcgtcgt gatcaaccac
      241 cacggcggca ctgaacaccg acaggcgcaa ctggtcgcgg ggctggcccc acgccacgcg
      301 gtcattgacc acgtaggccg acacggtgcc ggggccgttg agcttcacga cggagatcca
      361 gcgctcggcc accaagtcct tgactgcgta ttggaccgtc cgcaaagaac gtccgatgag
      421 cttggaaagt gtcttctggc tgaccaccac ggcgttctgg tggcccatct gcgccacgag
      481 gtgatgcagc agcattgccg ccgtgggttt cctcgcaata agcccggccc acgcctcatg
      541 cgctttgcgt tccgtttgca cccagtgacc gggcttgttc ttggcttgaa tgccgatttc
      601 tctggactgc gtggccatgc ttatctccat gcggtagggg tgccgcacgg ttgcggcacc
      661 atgcgcaatc agctgcaact tttcggcagc gcgacaacaa ttatgcgttg cgtaaaagtg
      721 gcagtcaatt acagattttc tttaacctac gcaatgagct attgcggggg gtgccgcaat
      781 gagctgttgc gtacccccct tttttaagtt gttgattttt aagtctttcg catttcgccc
      841 tatatctagt tctttggtgc ccaaagaagg gcacccctgc ggggttcccc cacgccttcg
      901 gcgcggctcc ccctccggca aaaagtggcc cctccggggc ttgttgatcg actgcgcggc
      961 cttcggcctt gcccaaggtg gcgctgcccc cttggaaccc ccgcactcgc cgccgtgagg
     1021 ctcggggggc aggcgggcgg gcttcgccct tcgactgccc ccactcgcat aggcttgggt
     1081 cgttccaggc gcgtcaaggc caagccgctg cgcggtcgct gcgcgagcct tgacccgcct
     1141 tccacttggt gtccaaccgg caagcgaagc gcgcaggccg caggccggag gcttttcccc
     1201 agagaaaatt aaaaaaattg atggggcaag gccgcaggcc gcgcagttgg agccggtggg
     1261 tatgtggtcg aaggctgggt agccggtggg caatccctgt ggtcaagctc gtgggcaggc
     1321 gcagcctgtc catcagcttg tccagcaggg ttgtccacgg gccgagcgaa gcgagccagc
     1381 cggtggccgc tcgcggccat cgtccacata tccacgggct ggcaagggag cgcagcgacc
     1441 gcgcagggcg aagcccggag agcaagcccg tagggcgccg cagccgccgt aggcggtcac
     1501 gactttgcga agcaaagtct agtgagtata ctcaagcatt gagtggcccg ccggaggcac
     1561 cgccttgcgc tgcccccgtc gagccggttg gacaccaaaa gggaggggca ggccagctgt
     1621 ctcttataca catctcgttg ttccatacaa cctccttagt acatgcaacc attatcaccg
     1681 ccagaggtaa aatagtcaac acgcacggtg ttagatattt atcccttgcg gtgatagatt
     1741 taacgtatga gcacaaaaaa gaaaccatta acacaagagc agcttgagga cgcacgtcgc
     1801 cttaaagcaa tttatgaaaa aaagaaaaat gaacttggct tatcccagga atctgtcgca
     1861 gacaagatgg ggatggggca gtcaggcgtt ggtgctttat ttaatggcat caatgcatta
     1921 aatgcttata acgccgcatt gcttacaaaa attctcaaag ttagcgttga agaatttagc
     1981 ccttcaatcg ccagagaaat ctacgagatg tatgaagcgg ttagtatgca gccgtcactt
     2041 agaagtgagt atgagtaccc tgttttttct catgttcagg cagggatgtt ctcacctgag
     2101 cttagaacct ttaccaaagg tgatgcggag agatgggtaa gcacaaccaa aaaagccagt
     2161 gattctgcat tctggcttga ggttgaaggt aattccatga ccgcaccaac aggctccaag
     2221 ccaagctttc ctgacggaat gttaattctc gttgaccctg agcaggctgt tgagccaggt
     2281 gatttctgca tagccagact tgggggtgat gagtttacct tcaagaaact gatcagggat
     2341 agcggtcagg tgtttttaca accactaaac ccacagtacc caatgatccc atgcaatgag
     2401 agttgttccg ttgtggggaa agttatcgct agtcagtggc ctgaagagac gtttggctga
     2461 tcggcaaggt gttctggtcg gcgcatagct gataacaatt gagcaagaat cttcatcgaa
     2521 ttaggggaat tttcactccc ctcagaacat aacatagtaa atggttgaat tcattcaaat
     2581 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2641 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2701 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2761 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2821 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2881 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2941 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3001 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3061 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3121 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3181 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3241 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3301 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3361 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3421 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3481 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3541 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3601 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3661 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3721 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tcttaacccg
     3781 gcctcagcgc cgggttttct ttgcctcacg atcgccccca aaacacataa ccaattgtat
     3841 ttattgaaaa ataaatagat acaactcact aaacatagca attcagatct ctcacctacc
     3901 aaacaatgcc cccctgcaaa aaataaattc atataaaaaa catacagata accatctgcg
     3961 gtgataaatt atctctggcg gtgttgacat aaataccact ggcggtgata ctgagcacat
     4021 cagcaggacg cactgaccac catgaaggtg acgctcttaa aaattaagcc ctgaagaagg
     4081 gcagcattca aagcagaagg ctttggggtg tgtgatacga aacgaagcat tggccgtaag
     4141 tgcgattccg gattactaat cagcccggca tttcgcgggc gatattttca cagctatttc
     4201 aggagttcag ccatgaacgc ttattacatt caggatcgtc ttgaggctca gagctgggcg
     4261 cgtcactacc agcagctcgc ccgtgaagag aaagaggcag aactggcaga cgacatggaa
     4321 aaaggcctgc cccagcacct gtttgaatcg ctatgcatcg atcatttgca acgccacggg
     4381 gccagcaaaa aatccattac ccgtgcgttt gatgacgatg ttgagtttca ggagcgcatg
     4441 gcagaacaca tccggtacat ggttgaaacc attgctcacc accaggttga tattgattca
     4501 gaggtataaa acgaatgagt actgcactcg caacgctggc tgggaagctg gctgaacgtg
     4561 tcggcatgga ttctgtcgac ccacaggaac tgatcaccac tcttcgccag acggcattta
     4621 aaggtgatgc cagcgatgcg cagttcatcg cattactgat cgttgccaac cagtacggcc
     4681 ttaatccgtg gacgaaagaa atttacgcct ttcctgataa gcagaatggc atcgttccgg
     4741 tggtgggcgt tgatggctgg tcccgcatca tcaatgaaaa ccagcagttt gatggcatgg
     4801 actttgagca ggacaatgaa tcctgtacat gccggattta ccgcaaggac cgtaatcatc
     4861 cgatctgcgt taccgaatgg atggatgaat gccgccgcga accattcaaa actcgcgaag
     4921 gcagagaaat cacggggccg tggcagtcgc atcccaaacg gatgttacgt cataaagcca
     4981 tgattcagtg tgcccgtctg gccttcggat ttgctggtat ctatgacaag gatgaagccg
     5041 agcgcattgt cgaaaatact gcatacactg cagaacgtca gccggaacgc gacatcactc
     5101 cggttaacga tgaaaccatg caggagatta acactctgct gatcgccctg gataaaacat
     5161 gggatgacga cttattgccg ctctgttccc agatatttcg ccgcgacatt cgtgcatcgt
     5221 cagaactgac acaggccgaa gcagtaaaag ctcttggatt cctgaaacag aaagccgcag
     5281 agcagaaggt ggcagcatga caccggacat tatcctgcag cgtaccggga tcgatgtgag
     5341 agctgtcgaa cagggggaag atgcgtggca caaattacgg ctcggcgtca tcaccgcttc
     5401 agaagttcac aacgtgatag caaaaccccg ctccggaaag aagtggcctg acatgaaaat
     5461 gtcctacttc cacaccctgc ttgctgaggt ttgcaccggt gtggctccgg aagttaacgc
     5521 taaagcactg gcctggggaa aacagtacga gaacgacgcc agaaccctgt ttgaatttac
     5581 ttccggcgtg aatgttactg aatccccgat catctatcgc gacgaaagta tgcgtaccgc
     5641 ctgctctccc gatggtttat gcagtgacgg caacggcctt gaactgaaat gcccgtttac
     5701 ctcccgggat ttcatgaagt tccggctcgg tggtttcgag gccataaagt cagcttacat
     5761 ggcccaggtg cagtacagca tgtgggtgac gcgaaaaaat gcctggtact ttgccaacta
     5821 tgacccgcgt atgaagcgtg aaggcctgca ttatgtcgtg attgagcggg atgaaaagta
     5881 catggcgagt tttgacgaga tcgtgccgga gttcatcgaa aaaatggacg aggcactggc
     5941 tgaaattggt tttgtatttg gggagcaatg gcgatgagag aggaggatta caaaatgcca
     6001 attcaggtct taccgccaca actggcgaac cagattgccg caggtgaggt ggtcgagcga
     6061 cctgcgtcgg tagtcaaaga actagtgaaa aacagcctcg atgcaggtgc gacgcgtatc
     6121 gatattgata tcgaacgcgg tggggcgaaa cttatccgca ttcgtgataa cggctgcggt
     6181 atcaaaaaag atgagctggc gctggcgctg gctcgtcatg ccaccagtaa aatcgcctct
     6241 ctggacgatc tcgaagccat tatcagcctg ggctttcgcg gtgaggcgct ggcgagtatc
     6301 agttcggttt cccgcctgac gctcacttca cgcaccgcag aacagcagga agcctggcag
     6361 gcctatgccg aagggcgcga tatgaacgtg acggtaaaac cggcggcgca tcctgtgggg
     6421 acgacgctgg aggtgctgga tctgttctac aacaccccgg cgcggcgcaa attcctgcgc
     6481 accgagaaaa ccgaatttaa ccacattgat gagatcatcc gccgcattgc gctggcgcgt
     6541 ttcgacgtca cgatcaacct gtcgcataac ggtaaaattg tgcgtcagta ccgcgcagtg
     6601 ccggaaggcg ggcaaaaaga acggcgctta ggcgcgattt gcggcaccgc ttttcttgaa
     6661 caagcgctgg cgattgaatg gcaacacggc gatctcacgc tacgcggctg ggtggccgat
     6721 ccaaatcaca ccacgcccgc actggcagaa attcagtatt gctacgtgaa cggtcgcatg
     6781 atgcgcgatc gcctgatcaa tcacgcgatc cgccaggcct gcgaagacaa actgggggcc
     6841 gatcagcaac cggcatttgt gttgtatctg gagatcgacc cacatcaggt ggacgtcaac
     6901 gtgcaccccg ccaaacacga agtgcgtttc catcagtcgc gtctggtgca tgattttatc
     6961 tatcagggcg tgctgagcgt gctacaacag caactggaaa cgccgctacc gctggacgat
     7021 gaaccccaac ctgcaccgcg ttccattccg gaaaaccgcg tggcggcggg gcgcaatcac
     7081 tttgcagaac cggcagctcg tgagccggta gctccgcgct acactcctgc gccagcatca
     7141 ggcagtcgtc cggctgcccc ctggccgaat gcgcagccag gctaccagaa acagcaaggt
     7201 gaagtgtatc gccagctttt gcaaacgccc gcgccgatgc aaaaattaaa agcgccggaa
     7261 ccgcaggaac ctgcacttgc ggcgaacagt cagagttttg gtcgggtact gactatcgtc
     7321 cattccgact gtgcgttgct ggagcgcgac ggcaacattt cacttttatc cttgccagtg
     7381 gcagaacgtt ggctgcgtca ggcacaattg acgccgggtg aagcgcccgt ttgcgcccag
     7441 ccgctgctga ttccgttgcg gctaaaagtt tctgccgaag aaaaatcggc attagaaaaa
     7501 gcgcagtctg ccctggcgga attgggtatt gatttccagt cagatgcaca gcatgtgacc
     7561 atcagggcag tgcctttacc cttacgccaa caaaatttac aaatcttgat tcctgaactg
     7621 ataggctacc tggcgaagca gtccgtattc gaacctggca atattgcgca gtggattgca
     7681 cgaaatctga tgagcgaaca tgcgcagtgg tcaatggcac aggccataac cctgctggcg
     7741 gacgtggaac ggttatgtcc gcaacttgtg aaaacgccgc cgggtggtct gttacaatct
     7801 gttgatttac atccggcgat aaaagccctg aaagatgagt gaaatggcga tgagcatcct
     7861 cacgataata tccgggtagg cgcaatcact ttcgtctact ccgttacaaa gcgaggctgg
     7921 gtatttcccg gcctttctgt tatccgaaat ccactgaaag cacagcggag atgtgtataa
     7981 gagacagctg cgaggcggta cagccgatag tctggaacag cgcacttacg ggttgctgcg
     8041 caacccaagt gctaccggcg cggcagcgtg acccgtgtcg gcggctccaa cggctc
//
