LOCUS pOSIP-KC (KanR, PhiC31) 6971 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 6971)
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 6971)
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-KC (KanR, PhiC31)
FEATURES Location/Qualifiers
source
1..6971
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 142..447
/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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
primer_bind
326..345
/label=ccdB-fwd
/note="ccdB gene,
forward primer"
rep_origin 477..1065
/direction=RIGHT
/label=ori
/note="high-copy-number
ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind
966..985
/label=pBR322ori-F
/note="pBR322
origin, forward primer"
misc_feature 1125..1145
/label=BioBrick suffix
/note="universal suffix for all parts"
terminator 1200..1227
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
terminator complement(1276..1307)
/label=tonB
terminator
/note="bidirectional E. coli tonB-P14 transcription
terminator"
protein_bind 1384..1417
/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(1424..2137)
/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 2226..2234
/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 2272..4089
/codon_start=1
/product="integrase from phage phi-C31"
/label=phage phi-C31 integrase
/translation="MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDG
GRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIP
IVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQREL
GGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWRE
IKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRIL
RDPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQA
WLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPG
QHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERA
NLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAE
APKLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPI
EKRASITWAKPPTDDDEDDAQDGTEDVAA"
primer_bind complement(4213..4232)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 4224..5039
/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
(4297..4316)
/label=Kan-R
/note="Kanamycin
resistance gene, reverse primer"
rep_origin 5161..5549
/label=R6K gamma ori
/note="gamma replication origin from E.
coli plasmid R6K;
requires the R6K initiator protein pi for
replication"
protein_bind 5560..5607
/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 5676..5775
/label=phage phi-C31 attP
/bound_moiety="phage phi-C31 integrase"
/note="attachment site of phage phi-C31"
terminator complement(5860..6106)
/label=lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
primer_bind
complement(6197..6219)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 6370..6388
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI
site, forward
primer"
terminator 6429..6515
/gene="Escherichia coli rrnB"
/label=rrnB T1
terminator
/note="transcription terminator T1 from the E. coli
rrnB
gene"
terminator 6612..6698
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 6795..6881
/gene="Escherichia coli
rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
ORIGIN
1 gaattcgagc tcggtacccg gggatcctct agagggctta ctaaaagcca gataacagta
61 tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag
121
tatgtcaaaa agaggtatgc tatgcagttt aaggtttaca cctataaaag agagagccgt
181
tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg
241
atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg
301
gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc
361
tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
421
attaacctga tgttctgggg aatataataa cccgtagaaa agatcaaaag atcttcttga
481
gatccttttt ttctgcgcgt aatctgctac ttgcaaacaa aaaaaccacc gctaccagcg
541
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggctttagc
601
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
661
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
721
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
781
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
841
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
901
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
961
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
1021
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
1081
gcctttttac ggttcctggc cttttgctgg ccttttgctc acattactag tagcggccgc
1141
tgcaggcatg cctcgagatg catggcgcct aacctaaact gacaggcatc aaattaagca
1201
gaaggccatc ctgacggatg gcctttttgc gtttcgaaca attgaaaaaa cctcgcgcct
1261
tacctgttga gtaatagtca aaagcctccg gtcggaggct tttgactttc tgcttactga
1321
atttcggtgg tgccgttaat taaccggtgg gccctcatga taataatggt ttcttagacg
1381
tccgaagttc ctattctcta gaaagtatag gaacttccct aggtcagcca aacgtctctt
1441
caggccactg actagcgata actttcccca caacggaaca actctcattg catgggatca
1501
ttgggtactg tgggtttagt ggttgtaaaa acacctgacc gctatccctg atcagtttct
1561
tgaaggtaaa ctcatcaccc ccaagtctgg ctatgcagaa atcacctggc tcaacagcct
1621
gctcagggtc aacgagaatt aacattccgt caggaaagct tggcttggag cctgttggtg
1681
cggtcatgga attaccttca acctcaagcc agaatgcaga atcactggct tttttggttg
1741
tgcttaccca tctctccgca tcacctttgg taaaggttct aagcttaggt gagaacatcc
1801
ctgcctgaac atgagaaaaa acagggtact catactcact tctaagtgac ggctgcatac
1861
taaccgcttc atacatctcg tagatttctc tggcgattga agggctaaat tcttcaacgc
1921
taactttgag aatttttgta agcaatgcgg cgttataagc atttaatgca ttgatgccat
1981
taaataaagc accaacgcct gactgcccca tccccatctt gtctgcgaca gattcctggg
2041
ataagccaag ttcatttttc tttttttcat aaattgcttt aaggcgacgt gcgtcctcaa
2101
gctgctcttg tgttaatggt ttcttttttg tgctcatacg ttaaatctat caccgcaagg
2161
gataaatatc taacaccgtg cgtgttgact attttacctc tggcggtgat aatggttgca
2221
tgtactaagg aggttgtatc ctgcaggccg agaaaaagga ggagataaac agtggacacg
2281
tacgcgggtg cttacgaccg tcagtcgcgc gagcgcgaaa attcgagcgc agcaagccca
2341
gcgacacagc gtagcgccaa cgaagacaag gcggccgacc ttcagcgcga agtcgagcgc
2401
gacgggggcc ggttcaggtt cgtcgggcat ttcagcgaag cgccgggcac gtcggcgttc
2461
gggacggcgg agcgcccgga gttcgaacgc atcctgaacg aatgccgcgc cgggcggctc
2521
aacatgatca ttgtctatga cgtgtcgcgc ttctcgcgcc tgaaggtcat ggacgcgatt
2581
ccgattgtct cggaattgct cgccctgggc gtgacgattg tttccactca ggaaggcgtc
2641
ttccggcagg gaaacgtcat ggacctgatt cacctgatta tgcggctcga cgcgtcgcac
2701
aaagaatctt cgctgaagtc ggcgaagatt ctcgacacga agaaccttca gcgcgaattg
2761
ggcgggtacg tcggcgggaa ggcgccttac ggcttcgagc ttgtttcgga gacgaaggag
2821
atcacgcgca acggccgaat ggtcaatgtc gtcatcaaca agcttgcgca ctcgaccact
2881
ccccttaccg gacccttcga gttcgagccc gacgtaatcc ggtggtggtg gcgtgagatc
2941
aagacgcaca aacaccttcc cttcaagccg ggcagtcaag ccgccattca cccgggcagc
3001
atcacggggc tttgtaagcg catggacgct gacgccgtgc cgacccgggg cgagacgatt
3061
gggaagaaga ccgcttcaag cgcctgggac ccggcaaccg ttatgcgaat ccttcgggac
3121
ccgcgtattg cgggcttcgc cgctgaggtg atctacaaga agaagccgga cggcacgccg
3181
accacgaaga ttgagggtta ccgcattcag cgcgacccga tcacgctccg gccggtcgag
3241
cttgattgcg gaccgatcat cgagcccgct gagtggtatg agcttcaggc gtggttggac
3301
ggcagggggc gcggcaaggg gctttcccgg gggcaagcca ttctgtccgc catggacaag
3361
ctgtactgcg agtgtggcgc cgtcatgact tcgaagcgcg gggaagaatc gatcaaggac
3421
tcttaccgct gccgtcgccg gaaggtggtc gacccgtccg cacctgggca gcacgaaggc
3481
acgtgcaacg tcagcatggc ggcactcgac aagttcgttg cggaacgcat cttcaacaag
3541
atcaggcacg ccgaaggcga cgaagagacg ttggcgcttc tgtgggaagc cgcccgacgc
3601
ttcggcaagc tcactgaggc gcctgagaag agcggcgaac gggcgaacct tgttgcggag
3661
cgcgccgacg ccctgaacgc ccttgaagag ctgtacgaag accgcgcggc aggcgcgtac
3721
gacggacccg ttggcaggaa gcacttccgg aagcaacagg cagcgctgac gctccggcag
3781
caaggggcgg aagagcggct tgccgaactt gaagccgccg aagccccgaa gcttcccctt
3841
gaccaatggt tccccgaaga cgccgacgct gacccgaccg gccctaagtc gtggtggggg
3901
cgcgcgtcag tagacgacaa gcgcgtgttc gtcgggctct tcgtagacaa gatcgttgtc
3961
acgaagtcga ctacgggcag ggggcaggga acgcccatcg agaagcgcgc ttcgatcacg
4021
tgggcgaagc cgccgaccga cgacgacgaa gacgacgccc aggacggcac ggaagacgta
4081
gcggcgtagt tttattttga ctaataatga cctacttaca cgttgtgtct caaaatctct
4141
gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
4201
taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctcccgtc
4261
cgcgcttaaa ctccaacatg gacgctgatt tatatgggta taaatgggct cgcgataatg
4321
tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
4381
ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtccgtctca
4441
actggctgac ggagtttatg cctctcccga ccatcaagca ttttatccgt actcctgatg
4501
atgcgtggtt actcaccacc gcgattcctg ggaaaacagc cttccaggta ttagaagaat
4561
atcctgattc aggtgaaaat attgttgatg cgctggccgt gttcctgcgc cggttacatt
4621
cgattcctgt ttgtaattgt ccttttaaca gcgatcgtgt atttcgtctt gctcaggcgc
4681
aatcacgcat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
4741
ggcctgttga acaagtctgg aaagaaatgc acaagctctt gccattctca ccggattcag
4801
tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
4861
gttgtattga tgttggacgg gtcggaatcg cagaccgtta ccaggacctt gccattcttt
4921
ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
4981
ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
5041
aatactagca gaaatcatcc ttagcgaaag ctaaggattt tttttatctg attaccgcct
5101
ttgagtgagc gtcgacctag tgcggccgca agatccggcc acgatgcgtc cggcgtagag
5161
gatctgaaga tcagcagttc aacctgttga tagtacgtac taagctctca tgtttcacgt
5221
actaagctct catgtttaac gtactaagct ctcatgttta acgaactaaa ccctcatggc
5281
taacgtacta agctctcatg gctaacgtac taagctctca tgtttcacgt actaagctct
5341
catgtttgaa caataaaatt aatataaatc agcaacttaa atagcctcta aggttttaag
5401
ttttataaga aaaaaaagaa tatataaggc ttttaaagct tttaaggttt aacggttgtg
5461
gacaacaagc cagggatgta acgcactgag aagcccttag agcctctcaa agcaattttg
5521
agtgacacag gaacacttaa cggctgacat gggaattagg aagttcctat tccgaagttc
5581
ctattctcta gaaagtatag gaacttccat atgccatggt cgcgctcgcg cgactgacgg
5641
tcgtaagcac ccgcgtacgt gtccaccccg gtcacaaccc cttgtgtcat gtcggcggcc
5701
ctacgccccc aactgagaga actcaaaggt taccccagtt ggggcactac tcccgaaaac
5761
cgcttctgac ctgggaaaac gtgaagcccc ggggcatccg ctgagggttg ccgccggggc
5821
ttcggtgtgt ccgtcagtag ctagcttctt cgtctgtttc tactggtatt ggcacaaacc
5881
tgattccaat ttgagcaagg ctatgtgcca tctcgatact cgttcttaac tcaacagaag
5941
atgctttgtg catacagccc ctcgtttatt atttatctcc tcagccagcc gctgtgcttt
6001
cagtggattt cggataacag aaaggccggg aaatacccag cctcgctttg taacggagta
6061
gagacgaaag tgattgcgcc tacccggata ttatcgtgag gatgcgtcat cgccattaat
6121
tcactgatca gtgataagct gtcaaacatg agaattgatc cggctgcctc gcgcgtttcg
6181
gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
6241
aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
6301
ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatgctgca catgacatta
6361
acctataaaa ataggcgtat cacgaggccc tttcgtcttc aagaattaat tcccaattcc
6421
ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
6481
gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
6541
cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggaattaat
6601
tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
6661
gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
6721
ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggaatt
6781
aattccccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
6841
tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
6901
acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccagga
6961
attggggatc g
//
质粒图谱.png)
