LOCUS       sequence-240243-        6248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrating mCherry into lambda attB in E. coli chromosome..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6248)
  AUTHORS   Cui L, Vigouroux A, Rousset F, Varet H, Khanna V, Bikard D
  TITLE     A CRISPRi screen in E. coli reveals sequence-specific toxicity of 
            dCas9.
  JOURNAL   Nat Commun. 2018 May 15;9(1):1912. doi: 10.1038/s41467-018-04209-5.
  PUBMED    29765036
REFERENCE   2  (bases 1 to 6248)
  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: "2018-05-15"; volume: "9(1):1912. doi"; pages: " 
            10.1038/s41467-018-04209-5"
COMMENT     Sequence Label: pOSIP-KL-mcherry
FEATURES             Location/Qualifiers
     source          1..6248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      60..87
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(136..167)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     protein_bind    244..277
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(283..996)
                     /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
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     RBS             1085..1093
                     /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             1238..2308
                     /codon_start=1
                     /gene="int"
                     /product="integrase from phage lambda"
                     /label=lambda integrase
                     /translation="MGRRRSHERRDLPPNLYIRNNGYYCYRDPRTGKEFGLGRDRRIAI
                     TEAIQANIELFSGHKHKPLTARINSDNSVTLHSWLDRYEKILASRGIKQKTLINYMSKI
                     KAIRRGLPDAPLEDITTKEIAAMLNGYIDEGKAASAKLIRSTLSDAFREAIAEGHITTN
                     HVAATRAAKSEVRRSRLTADEYLKIYQAAESSPCWLRLAMELAVVTGQRVGDLCEMKWS
                     DIVDGYLYVEQSKTGVKIAIPTALHIDALGISMKETLDKCKEILGGETIIASTRREPLS
                     SGTVSRYFMRARKASGLSFEGDPPTFHELRSLSARLYEKQISDKFAQHLLGHKSDTMAS
                     QYRDDRGREWDKIEIK"
     primer_bind     complement(2524..2543)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2535..3350
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2608..2627)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3472..3860
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    3871..3918
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    3981..4214
                     /gene="attP"
                     /label=lambda attP
                     /bound_moiety="lambda integrase"
                     /note="integrase from phage lambda"
     terminator      complement(4338..4584)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(4675..4697)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4848..4866
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      4907..4993
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5090..5176
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5273..5359
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tgcaggcatg cctcgagatg catggcgcct aacctaaact gacaggcatc aaattaagca
       61 gaaggccatc ctgacggatg gcctttttgc gtttcgaaca attgaaaaaa cctcgcgcct
      121 tacctgttga gtaatagtca aaagcctccg gtcggaggct tttgactttc tgcttactga
      181 atttcggtgg tgccgttaat taaccggtgg gccctcatga taataatggt ttcttagacg
      241 tccgaagttc ctattctcta gaaagtatag gaacttccct agtcagccaa acgtctcttc
      301 aggccactga ctagcgataa ctttccccac aacggaacaa ctctcattgc atgggatcat
      361 tgggtactgt gggtttagtg gttgtaaaaa cacctgaccg ctatccctga tcagtttctt
      421 gaaggtaaac tcatcacccc caagtctggc tatgcagaaa tcacctggct caacagcctg
      481 ctcagggtca acgagaatta acattccgtc aggaaagctt ggcttggagc ctgttggtgc
      541 ggtcatggaa ttaccttcaa cctcaagcca gaatgcagaa tcactggctt ttttggttgt
      601 gcttacccat ctctccgcat cacctttggt aaaggttcta agcttaggtg agaacatccc
      661 tgcctgaaca tgagaaaaaa cagggtactc atactcactt ctaagtgacg gctgcatact
      721 aaccgcttca tacatctcgt agatttctct ggcgattgaa gggctaaatt cttcaacgct
      781 aactttgaga atttttgtaa gcaatgcggc gttataagca tttaatgcat tgatgccatt
      841 aaataaagca ccaacgcctg actgccccat ccccatcttg tctgcgacag attcctggga
      901 taagccaagt tcatttttct ttttttcata aattgcttta aggcgacgtg cgtcctcaag
      961 ctgctcttgt gttaatggtt tcttttttgt gctcatacgt taaatctatc accgcaaggg
     1021 ataaatatct aacaccgtgc gtgttgacta ttttacctct ggcggtgata atggttgcat
     1081 gtactaagga ggttgtatac gcgtcgtcga tgggttcggg aatgcaggat attcccacct
     1141 ccggttaagg atggaagaga gtatctgttc cacgaatcag cggtaaaggt tgacttaaat
     1201 cgaccagtaa caggtggcct tttgaagagg atcagaaatg ggaagaaggc gaagtcatga
     1261 gcgccgggat ttacccccta acctttatat aagaaacaat ggatattact gctacaggga
     1321 cccaaggacg ggtaaagagt ttggattagg cagagacagg cgaatcgcaa tcactgaagc
     1381 tatacaggcc aacattgagt tattttcagg acacaaacac aagcctctga cagcgagaat
     1441 caacagtgat aattccgtta cgttacattc atggcttgat cgctacgaaa aaatcctggc
     1501 cagcagagga atcaagcaga agacactcat aaattacatg agcaaaatta aagcaataag
     1561 gaggggtctg cctgatgctc cacttgaaga catcaccaca aaagaaattg cggcaatgct
     1621 caatggatac atagacgagg gcaaggcggc gtcagccaag ttaatcagat caacactgag
     1681 cgatgcattc cgagaggcaa tagctgaagg ccatataaca acaaaccatg tcgctgccac
     1741 tcgcgcagca aaatcagagg taaggagatc aagacttacg gctgacgaat acctgaaaat
     1801 ttatcaagca gcagaatcat caccatgttg gctcagactt gcaatggaac tggctgttgt
     1861 taccgggcaa cgagttggtg atttatgcga aatgaagtgg tctgatatcg tagatggata
     1921 tctttatgtc gagcaaagca aaacaggcgt aaaaattgcc atcccaacag cattgcatat
     1981 tgatgctctc ggaatatcaa tgaaggaaac acttgataaa tgcaaagaga ttcttggcgg
     2041 agaaaccata attgcatcta ctcgtcgcga accgctttca tccggcacag tatcaaggta
     2101 ttttatgcgc gcacgaaaag catcaggtct ttccttcgaa ggggatccgc ctacctttca
     2161 cgagttgcgc agtttgtctg caagactcta tgagaagcag ataagcgata agtttgctca
     2221 acatcttctc gggcataagt cggacaccat ggcatcacag tatcgtgatg acagaggcag
     2281 ggagtgggac aaaattgaaa tcaaataatg attttatttt gactgatagt gacctgttcg
     2341 ttgcaacaaa ttgataagca atgccaagct tggcactggc tgatcagcta gcccatgggt
     2401 atggacagtt ttccctttga tatgtaacgc acgttgtgtc tcaaaatctc tgatgttaca
     2461 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     2521 atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctcccgt ccgcgcttaa
     2581 actccaacat ggacgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     2641 caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac
     2701 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtccgtctc aactggctga
     2761 cggagtttat gcctctcccg accatcaagc attttatccg tactcctgat gatgcgtggt
     2821 tactcaccac cgcgattcct gggaaaacag ccttccaggt attagaagaa tatcctgatt
     2881 caggtgaaaa tattgttgat gcgctggccg tgttcctgcg ccggttacat tcgattcctg
     2941 tttgtaattg tccttttaac agcgatcgtg tatttcgtct tgctcaggcg caatcacgca
     3001 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     3061 aacaagtctg gaaagaaatg cacaagctct tgccattctc accggattca gtcgtcactc
     3121 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     3181 atgttggacg ggtcggaatc gcagaccgtt accaggacct tgccattctt tggaactgcc
     3241 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     3301 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa taatactagc
     3361 agaaatcatc cttagcgaaa gctaaggatt ttttttatct gattaccgcc tttgagtgag
     3421 cgtcgaccta gtgcggccgc aagatccggc cacgatgcgt ccggcgtaga ggatctgaag
     3481 atcagcagtt caacctgttg atagtacgta ctaagctctc atgtttcacg tactaagctc
     3541 tcatgtttaa cgtactaagc tctcatgttt aacgaactaa accctcatgg ctaacgtact
     3601 aagctctcat ggctaacgta ctaagctctc atgtttcacg tactaagctc tcatgtttga
     3661 acaataaaat taatataaat cagcaactta aatagcctct aaggttttaa gttttataag
     3721 aaaaaaaaga atatataagg cttttaaagc ttttaaggtt taacggttgt ggacaacaag
     3781 ccagggatgt aacgcactga gaagccctta gagcctctca aagcaatttt gagtgacaca
     3841 ggaacactta acggctgaca tgggaattag gaagttccta ttccgaagtt cctattctct
     3901 agaaagtata ggaacttcca tatgccatgg catcacagta tcgtgatgac agaggcaggg
     3961 agtgggacaa aattgaaatc aaataatgat tttattttga ctgatagtga cctgttcgtt
     4021 gcaacaaatt gataagcaat gcttttttat aatgccaact tagtataaaa aagctgaacg
     4081 agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata aaaaacagac
     4141 tacataatac tgtaaaacac aacatatatg cagtcactat gaatcaacta cttagatggt
     4201 attagtgacc tgtaacagag cattagcgca aggtgatttt tgtcttcttg cgctaatttt
     4261 ttgtcatcaa acctgtcgca ctccagagaa gcacaaagcc tcgcaatcca gtgcaaagct
     4321 agcttcttcg tctgtttcta ctggtattgg cacaaacctg attccaattt gagcaaggct
     4381 atgtgccatc tcgatactcg ttcttaactc aacagaagat gctttgtgca tacagcccct
     4441 cgtttattat ttatctcctc agccagccgc tgtgctttca gtggatttcg gataacagaa
     4501 aggccgggaa atacccagcc tcgctttgta acggagtaga gacgaaagtg attgcgccta
     4561 cccggatatt atcgtgagga tgcgtcatcg ccattaattc actgatcagt gataagctgt
     4621 caaacatgag aattgatccg gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     4681 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     4741 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     4801 acgtagcgat agcggagtgt atgctgcaca tgacattaac ctataaaaat aggcgtatca
     4861 cgaggccctt tcgtcttcaa gaattaattc ccaattcccc aggcatcaaa taaaacgaaa
     4921 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     4981 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     5041 gcgggcagga cgcccgccat aaactgccag gaattaattc cccaggcatc aaataaaacg
     5101 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     5161 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg
     5221 gtggcgggca ggacgcccgc cataaactgc caggaattaa ttccccaggc atcaaataaa
     5281 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     5341 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg
     5401 agggtggcgg gcaggacgcc cgccataaac tgccaggaat tggggatcgg cctatgaact
     5461 gtcgactcga ggccttgaca ggtacctcat ggatacctat aatggttccg gaattccttt
     5521 aaagagggga aatctagatg gtttccaagg gcgaggagga taacatggct atcattaaag
     5581 agttcatgcg cttcaaagtt cacatggagg gttctgttaa cggtcacgag ttcgagatcg
     5641 aaggcgaagg cgagggccgt ccgtatgaag gcacccagac cgccaaactg aaagtgacta
     5701 aaggcggccc gctgcctttt gcgtgggaca tcctgagccc gcaatttatg tacggttcta
     5761 aagcttatgt taaacaccca gcggatatcc cggactatct gaagctgtct tttccggaag
     5821 gtttcaagtg ggaacgcgta atgaattttg aagatggtgg tgtcgtgacc gtcactcagg
     5881 actcctccct gcaggatggc gagttcatct ataaagttaa actgcgtggt actaattttc
     5941 catctgatgg cccggtgatg cagaagaaga cgatgggttg ggaggcgtct agcgaacgca
     6001 tgtatccgga agatggtgcg ctgaaaggcg aaattaaaca gcgcctgaaa ctgaaagatg
     6061 gcggccatta tgacgctgaa gtgaaaacca cgtacaaagc caagaaacct gtgcagctgc
     6121 ctggcgcgta caatgtgaat attaaactgg acatcacctc tcataatgaa gattatacga
     6181 tcgtagagca atatgagcgc gcggagggtc gtcattctac cggtggcatg gatgagctgt
     6241 acaaataa
//
