LOCUS pOSIP-KL-mcherry 6248 bp DNA circular SYN 14-APR-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 Apr 14, 2025 from SnapGene Server 8.0.1
https://www.biofeng.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
//
