LOCUS pOSIP-KP (KanR, Phi80) 6763 bp DNA circular SYN 14-APR-2025
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6763)
AUTHORS St-Pierre F, Cui L, Priest DG, Endy D, Dodd IB, Shearwin KE
TITLE One-step cloning and chromosomal integration of DNA.
JOURNAL ACS Synth Biol. 2013 Sep 20;2(9):537-41. doi: 10.1021/sb400021j.
Epub 2013 May 20.
PUBMED 24050148
REFERENCE 2 (bases 1 to 6763)
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"; doi: "10.1021/sb400021j";
journalName: "ACS Synth Biol"; date: "2013-09-20- 20"; volume: "2";
issue: "9"; pages: "537-41"
COMMENT Sequence Label: pOSIP-KP (KanR, Phi80)
FEATURES Location/Qualifiers
source 1..6763
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 151..456
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRQMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
primer_bind 335..354
/label=ccdB-fwd
/note="ccdB gene, forward primer"
rep_origin 486..1074
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 975..994
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
misc_feature 1134..1154
/label=BioBrick suffix
/note="universal suffix for all parts"
terminator 1203..1230
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
terminator complement(1279..1310)
/label=tonB terminator
/note="bidirectional E. coli tonB-P14 transcription
terminator"
protein_bind 1387..1420
/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(1427..2140)
/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 2229..2237
/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)"
primer_bind complement(3745..3764)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 3756..4571
/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(3829..3848)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
rep_origin 4693..5081
/label=R6K gamma ori
/note="gamma replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication"
protein_bind 5092..5139
/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 5246..5563
/label=phage Phi-80 attP
/bound_moiety="phage phi-80 integrase"
/note="attachment site of phage phi-80"
terminator complement(5652..5898)
/label=lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
primer_bind complement(5989..6011)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 6162..6180
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
terminator 6221..6307
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 6404..6490
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 6587..6673
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
ORIGIN
1 gaattcgagc tcggtacccg gggatccgcg gccgcttcta gagggcttac taaaagccag
61 ataacagtat gcgtacttgc gcgctgattt ttgcggtata agaatatata ctgatatgta
121 tacccgaagt atgtcaaaaa gaggtatgct atgcagttta aggtttacac ctataaaaga
181 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga
241 cagatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt
301 tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt
361 gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
421 aaaaacgcca ttaacctgat gttctgggga atataataac ccgtagaaaa gattaaaaga
481 tcttcttgag atcctttttt tctgcgcgta atctgctact tgcaaacaaa aaaaccaccg
541 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
601 ggctttagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
661 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
721 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
781 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
841 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
901 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
961 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
1021 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
1081 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca cattactagt
1141 agcggccgct gcagctcgag atgcatggcg cctaacctaa actgacaggc atcaaattaa
1201 gcagaaggcc atcctgacgg atggcctttt tgcgtttcga acaattgaaa aaacctcgcg
1261 ccttacctgt tgagtaatag tcaaaagcct ccggtcggag gcttttgact ttctgcttac
1321 tgaatttcgg tggtgccgtt aattaaccgg tgggccctca tgataataat ggtttcttag
1381 acgtccgaag ttcctattct ctagaaagta taggaacttc cctaggtcag ccaaacgtct
1441 cttcaggcca ctgactagcg ataactttcc ccacaacgga acaactctca ttgcatggga
1501 tcattgggta ctgtgggttt agtggttgta aaaacacctg accgctatcc ctgatcagtt
1561 tcttgaaggt aaactcatca cccccaagtc tggctatgca gaaatcacct ggctcaacag
1621 cctgctcagg gtcaacgaga attaacattc cgtcaggaaa gcttggcttg gagcctgttg
1681 gtgcggtcat ggaattacct tcaacctcaa gccagaatgc agaatcactg gcttttttgg
1741 ttgtgcttac ccatctctcc gcatcacctt tggtaaaggt tctaagctta ggtgagaaca
1801 tccctgcctg aacatgagaa aaaacagggt actcatactc acttctaagt gacggctgca
1861 tactaaccgc ttcatacatc tcgtagattt ctctggcgat tgaagggcta aattcttcaa
1921 cgctaacttt gagaattttt gtaagcaatg cggcgttata agcatttaat gcattgatgc
1981 cattaaataa agcaccaacg cctgactgcc ccatccccat cttgtctgcg acagattcct
2041 gggataagcc aagttcattt ttcttttttt cataaattgc tttaaggcga cgtgcgtcct
2101 caagctgctc ttgtgttaat ggtttctttt ttgtgctcat acgttaaatc tatcaccgca
2161 agggataaat atctaacacc gtgcgtgttg actattttac ctctggcggt gataatggtt
2221 gcatgtacta aggaggttgt atacgcgtta gcaaccctta tccagcaggc tttcaaggat
2281 gtaaaccata acactctgcg aactagtgtt acattgcgtg tagctttgag tgggcaactt
2341 tgtgtacact tttgtgtacc caaaaacaaa aatgtgtacc cattcaatga tcaccgacac
2401 aaagctcagg aagggctcgg caagaaaaga gatgatatcg agattatttc tgattcgcac
2461 gggctcaacg ccagaatcag ccaggccgga aaaatatcat ttttctatcg gtatcgctgg
2521 gccggtaaag cggtaaaact caatgttggt gattatcctg caatgagtat cacccaggca
2581 agagagcgtc gccaacaatt cagaaactgg ttaactgagg gactggatcc gcgagagcag
2641 gtgaagctgg ataagcagac ccgacaggaa gcgatgtccg ttgccgaagc gttcaattac
2701 tggattgaaa ggcactgtat cgctaacggg ctagttaaag tcgattacta tcgccaggtg
2761 tttgagaaac atatcgccga accgatgaag aatgtcaaag tcgataacac agcgaaaatg
2821 cactggatca acgtcttcga ttctatagaa agcagggtga tggctcatta catgctttcg
2881 ctgtgcaaac gggcgtttag gttctgcgtt aacagaagtg tgatcgcctc aaacccactc
2941 gagggattac tgccatctga tgtcgggcaa aagcctaaaa agagaactcg caggatggac
3001 gatgacgatc tgcgcaaaat ctatcagtgg ttgaaaagcc atatgtcgat agagtccgtt
3061 ttcctggtga aatttattat gcttaccgga tgccgtacgg ctgagattcg acttagtgag
3121 agatcatggt ttcgattgga tgataatgag tgggtcgtgc ctgcgggcag ttataaaact
3181 cgggtacata ttagaagggg actctcagac gccgccgtta acctggtcag aaatcacctc
3241 aagaaaataa acaccaatca cctggtgact tcacaacgta aaattgatgg cgggatcaaa
3301 gattcgcccg ttcattcacc tgtggcatcc aattacgccc gttctatttg gaatggaaca
3361 ggtatggcag agtggtcgct tcatgatatg aggcggacga tagccacaaa tctctctgag
3421 ttaggttgcc cgccgcacgt aattgaaaag ctgctcgggc atcagatggt gggggttatg
3481 gcgcattaca accttcatga ctatatcgat gatcagaaac actggctccg cgtttggcag
3541 agccatcttg aagagatcat cggagagccc ttcagttaat ttatcttctt tttatcctcc
3601 cactctttga ttgccatggg tatggacagt tttccctttg atatgtaacg cacgttgtgt
3661 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
3721 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
3781 cttgctcccg tccgcgctta aactccaaca tggacgctga tttatatggg tataaatggg
3841 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg
3901 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
3961 tggtccgtct caactggctg acggagttta tgcctctccc gaccatcaag cattttatcc
4021 gtactcctga tgatgcgtgg ttactcacca ccgcgattcc tgggaaaaca gccttccagg
4081 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggcc gtgttcctgc
4141 gccggttaca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgt gtatttcgtc
4201 ttgctcaggc gcaatcacgc atgaataacg gtttggttga tgcgagtgat tttgatgacg
4261 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcacaagctc ttgccattct
4321 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
4381 ggaaattaat aggttgtatt gatgttggac gggtcggaat cgcagaccgt taccaggacc
4441 ttgccattct ttggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
4501 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
4561 agtttttcta ataatactag cagaaatcat ccttagcgaa agctaaggat tttttttatc
4621 tgattaccgc ctttgagtga gcgtcgacct agtgcggccg caagatccgg ccacgatgcg
4681 tccggcgtag aggatctgaa gatcagcagt tcaacctgtt gatagtacgt actaagctct
4741 catgtttcac gtactaagct ctcatgttta acgtactaag ctctcatgtt taacgaacta
4801 aaccctcatg gctaacgtac taagctctca tggctaacgt actaagctct catgtttcac
4861 gtactaagct ctcatgtttg aacaataaaa ttaatataaa tcagcaactt aaatagcctc
4921 taaggtttta agttttataa gaaaaaaaag aatatataag gcttttaaag cttttaaggt
4981 ttaacggttg tggacaacaa gccagggatg taacgcactg agaagccctt agagcctctc
5041 aaagcaattt tgagtgacac aggaacactt aacggctgac atgggaatta ggaagttcct
5101 attccgaagt tcctattctc tagaaagtat aggaacttcc atatgccatg ggcccgtgcg
5161 aatcagaaat aatctcgata tcatctcttt tcttgccgag cgccttcctg agctttgtgt
5221 cggtgatcat tgaatgggta cacatttttg tttttgggta cacaaaagtg tacacaaagt
5281 tgcccactca aagctacacg caatgtaaca ctagttcgca gagtgttatg gtttacatcc
5341 ttgaaagcct gctggataag ggtttagcgt aacagaacgt ttttacgcgg aattgttcgt
5401 aatatgccaa atgacaattt aagaaagtgt tctaatttat tagaaatttg cacttaaatc
5461 aaaaagttac ggacaattca accaccaatc aataaattaa agggcacatt aaagtacaca
5521 atatttgtgc ccttctctgt ttctttcccg ttatcagcta gctggaaact ttttatacag
5581 agtttagagc cctactccat acgtttttga tacgctttgt cgtgattcac ctctagcttc
5641 ttcgtctgtt tctactggta ttggcacaaa cctgattcca atttgagcaa ggctatgtgc
5701 catctcgata ctcgttctta actcaacaga agatgctttg tgcatacagc ccctcgttta
5761 ttatttatct cctcagccag ccgctgtgct ttcagtggat ttcggataac agaaaggccg
5821 ggaaataccc agcctcgctt tgtaacggag tagagacgaa agtgattgcg cctacccgga
5881 tattatcgtg aggatgcgtc atcgccatta attcactgat cagtgataag ctgtcaaaca
5941 tgagaattga tccggctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
6001 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
6061 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
6121 cgatagcgga gtgtatgctg cacatgacat taacctataa aaataggcgt atcacgaggc
6181 cctttcgtct tcaagaatta attcccaatt ccccaggcat caaataaaac gaaaggctca
6241 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
6301 gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc
6361 aggacgcccg ccataaactg ccaggaatta attccccagg catcaaataa aacgaaaggc
6421 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
6481 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
6541 ggcaggacgc ccgccataaa ctgccaggaa ttaattcccc aggcatcaaa taaaacgaaa
6601 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
6661 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
6721 gcgggcagga cgcccgccat aaactgccag gaattgggga tcg
//
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