LOCUS pOSIP-KT (KanR, P21) 6587 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 6587)
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 6587)
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-KT (KanR, P21)
FEATURES Location/Qualifiers
source 1..6587
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator complement(60..146)
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
primer_bind complement(187..205)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind 356..378
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
terminator 469..715
/label=lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
protein_bind 736..1235
/label=phage phi-21 attP
/bound_moiety="phage phi-21 integrase"
/note="attachment site of phage phi-21"
protein_bind complement(1315..1362)
/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)."
rep_origin 1373..1761
/label=R6K gamma ori
/note="gamma replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication"
CDS complement(1883..2698)
/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 2606..2625
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 2690..2709
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS complement(2933..4075)
/codon_start=1
/gene="int"
/product="integrase from phage phi-21"
/label=phage phi-21 integrase
/translation="MAARPRSHKISIPNLYCKLDKRTGKVYWQYKHPLSGRFHSLGTDE
NEAKQVATEANTIIAEQRTRQILSVNERLERMKGRRSDITVTEWLDKYISIQEDRLQHN
ELRPNSYRQKGKPIRLFREHCGMQHLKDITALDIAEIIDAVKAEGHNRMAQVVRMVLID
VFKEAQHAGHVPPGFNPAQATKQPRNRVNRQRLSLLEWQAIFESVSRRQPYLKCGMLLA
LVTGQRLGDICNLKFSDIWDDMLHITQEKTGSKLAIPLNLKCDALNITLREVISQCRDA
VVSKYLVHYRHTTSQANRGDQVSANTLTTAFKKAKEKCGIKWEQGTAPTFHEQRSLSER
LYREQGLDTQKLLGHKSRKMTDRYNDDRGKDWIIVDIKTA"
RBS 4140..4148
/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 4237..4950
/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"
protein_bind complement(4957..4990)
/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)"
terminator 5067..5098
/label=tonB terminator
/note="bidirectional E. coli tonB-P14 transcription
terminator"
terminator complement(5147..5174)
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
misc_feature 5229..5249
/label=BioBrick suffix
/note="universal suffix for all parts"
rep_origin complement(5309..5897)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5389..5408)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(5927..6232)
/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 complement(6029..6048)
/label=ccdB-fwd
/note="ccdB gene, forward primer"
terminator complement(6464..6550)
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
ORIGIN
1 cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt tcaaatccgc tcccggcgga
61 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
121 ttcgactgag cctttcgttt tatttgatgc ctggggaatt gggaattaat tcttgaagac
181 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgtgcagc atacactccg
241 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
301 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
361 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gccggatcaa
421 ttctcatgtt tgacagctta tcactgatca gtgaattaat ggcgatgacg catcctcacg
481 ataatatccg ggtaggcgca atcactttcg tctctactcc gttacaaagc gaggctgggt
541 atttcccggc ctttctgtta tccgaaatcc actgaaagca cagcggctgg ctgaggagat
601 aaataataaa cgaggggctg tatgcacaaa gcatcttctg ttgagttaag aacgagtatc
661 gagatggcac atagccttgc tcaaattgga atcaggtttg tgccaatacc agtagaaaca
721 gacgaagaag ctagcataag gcctcgcaat ggcttgcaag gccacacatg tattgagatg
781 ttaataaaat gtagacttgt aattttgata taaatggtag agaaaatctt tccccaaaat
841 aaaaacgaac gtcaatgaaa tcaaacggtt gaataaagtt gattttggct aatacaaaga
901 caagaaaata atatttatga ttaaatatca gcgagttgaa tacataattt ttatatactg
961 ctgcgccata tgggctggac tgaagccgca gacctgattg ttaaaggtat ggaaggcgca
1021 atcaacgcca agaccgtaac ctatgacttc gaacgtctga tggaaggcgc taaactgctg
1081 aaatgcagcg agtttggtga cgcgatcatc aaaaatatgt aattaccaca tgtgttaaat
1141 attataacgg gcgtataaca cgcccgttgt tttatgatga tgtaaaatct tccccaaaac
1201 tttccccaaa acccttcccc aaaactggct attttctatg ctgttttgat atctacgata
1261 atccagtctt taccacgatc atcattgtat cggtcggtca ttccatggca tatggaagtt
1321 cctatacttt ctagagaata ggaacttcgg aataggaact tcctaattcc catgtcagcc
1381 gttaagtgtt cctgtgtcac tcaaaattgc tttgagaggc tctaagggct tctcagtgcg
1441 ttacatccct ggcttgttgt ccacaaccgt taaaccttaa aagctttaaa agccttatat
1501 attctttttt ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat
1561 taattttatt gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacgttag
1621 ccatgagagc ttagtacgtt agccatgagg gtttagttcg ttaaacatga gagcttagta
1681 cgttaaacat gagagcttag tacgtgaaac atgagagctt agtacgtact atcaacaggt
1741 tgaactgctg atcttcagat cctctacgcc ggacgcatcg tggccggatc ttgcggccgc
1801 actaggtcga cgctcactca aaggcggtaa tcagataaaa aaaatcctta gctttcgcta
1861 aggatgattt ctgctagtat tattagaaaa actcatcgag catcaaatga aactgcaatt
1921 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
1981 aaaactcacc gaggcagttc caaagaatgg caaggtcctg gtaacggtct gcgattccga
2041 cccgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
2101 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caagagcttg tgcatttctt
2161 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
2221 aaccgttatt catgcgtgat tgcgcctgag caagacgaaa tacacgatcg ctgttaaaag
2281 gacaattaca aacaggaatc gaatgtaacc ggcgcaggaa cacggccagc gcatcaacaa
2341 tattttcacc tgaatcagga tattcttcta atacctggaa ggctgttttc ccaggaatcg
2401 cggtggtgag taaccacgca tcatcaggag tacggataaa atgcttgatg gtcgggagag
2461 gcataaactc cgtcagccag ttgagacgga ccatctcatc tgtaacatca ttggcaacgc
2521 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aagcgataga
2581 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcgt
2641 ccatgttgga gtttaagcgc ggacgggagc aagacgtttc ccgttgaata tggctcataa
2701 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt
2761 tatcttgtgc aatgtaacat cagagatttt gagacacaac gtgcgttaca tatcaaaggg
2821 aaaactgtcc atacccatgg ataacgggcg tataacacgc ccgttgtttt atgatgatgt
2881 aaaatcttcc ccaaaacttt ccccaaaacc cttccccaaa actggctatt ttctatgctg
2941 ttttgatatc tacgataatc cagtctttac cacgatcatc attgtatcgg tcggtcattt
3001 ttctggattt atggcctaac aacttttgcg tatccagacc ctgttcccga tataatcgtt
3061 ctgacagaga tcgctgttca tgaaatgtgg gcgcagttcc ttgctcccat tttatgccac
3121 atttttcctt ggccttttta aaagccgttg tcagagtatt tgcagaaacc tggtctcctc
3181 tgtttgcttg agaggtagtg tgacggtaat ggaccagata tttactaaca acagcatccc
3241 tgcactgaga tataacttca cgaagggtaa tattcagagc atcgcatttc aggttaagcg
3301 gaatagcaag ttttgaaccg gttttttcct gagtaatgtg caacatgtcg tcccatatat
3361 cagagaattt caaattgcag atatcgccta aacgttgtcc agtaacaaga gcaagtagca
3421 tgccgcattt taaatagggc tgccgtctgc ttacgctttc aaatattgcc tgccattcga
3481 gcagtgacaa tctttggcgg tttactcgat ttcgcggttg ttttgttgcc tgcgctgggt
3541 taaatcctgg cggaacatgt cctgcgtgtt gtgcttcttt gaagacgtcg atcaacacca
3601 ttctcacgac ttgcgccatc ctgttatgac cttcagcctt tacagcatca attatttcgg
3661 caatatcaag tgcggtaata tccttgaggt gttgcattcc acaatgctca cggaaaagac
3721 gaatgggttt gcctttttgc cgataggagt tgggtcttag ttcattatgt tgcagcctgt
3781 cctcctggat agaaatatat ttatcaagcc attctgtcac cgtaatgtct gagcgcctgc
3841 ctttcattct ttccagacgc tcattgacgc ttaatatttg tctggtacgt tgttcagcaa
3901 taatggtatt tgcttcagta gcaacttgtt ttgcttcatt ctcatcagtt cctaagctat
3961 gaaaacgacc ggatagtgga tgtttgtatt gccaatatac ctttccggtt cgcttatcta
4021 atttgcaata taaattgggt atagagattt tgtgagatcg gggtctagca gccatcagcg
4081 attatccgtt ggagttttgg gtttgcgttt attgggagtt gcggttctac gcgtatacaa
4141 cctccttagt acatgcaacc attatcaccg ccagaggtaa aatagtcaac acgcacggtg
4201 ttagatattt atcccttgcg gtgatagatt taacgtatga gcacaaaaaa gaaaccatta
4261 acacaagagc agcttgagga cgcacgtcgc cttaaagcaa tttatgaaaa aaagaaaaat
4321 gaacttggct tatcccagga atctgtcgca gacaagatgg ggatggggca gtcaggcgtt
4381 ggtgctttat ttaatggcat caatgcatta aatgcttata acgccgcatt gcttacaaaa
4441 attctcaaag ttagcgttga agaatttagc ccttcaatcg ccagagaaat ctacgagatg
4501 tatgaagcgg ttagtatgca gccgtcactt agaagtgagt atgagtaccc tgttttttct
4561 catgttcagg cagggatgtt ctcacctaag cttagaacct ttaccaaagg tgatgcggag
4621 agatgggtaa gcacaaccaa aaaagccagt gattctgcat tctggcttga ggttgaaggt
4681 aattccatga ccgcaccaac aggctccaag ccaagctttc ctgacggaat gttaattctc
4741 gttgaccctg agcaggctgt tgagccaggt gatttctgca tagccagact tgggggtgat
4801 gagtttacct tcaagaaact gatcagggat agcggtcagg tgtttttaca accactaaac
4861 ccacagtacc caatgatccc atgcaatgag agttgttccg ttgtggggaa agttatcgct
4921 agtcagtggc ctgaagagac gtttggctga cctagggaag ttcctatact ttctagagaa
4981 taggaacttc ggacgtctaa gaaaccatta ttatcatgag ggcccaccgg ttaattaacg
5041 gcaccaccga aattcagtaa gcagaaagtc aaaagcctcc gaccggaggc ttttgactat
5101 tactcaacag gtaaggcgcg aggttttttc aattgttcga aacgcaaaaa ggccatccgt
5161 caggatggcc ttctgcttaa tttgatgcct gtcagtttag gttaggcgcc atgcatctcg
5221 aggcatgcct gcagcggccg ctactagtaa tgtgagcaaa aggccagcaa aaggccagga
5281 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
5341 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
5401 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
5461 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
5521 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
5581 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
5641 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
5701 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
5761 gtatctgcgc tctgctaaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
5821 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagtagcag attacgcgca
5881 gaaaaaaagg atctcaagaa gatcttttga tcttttctac gggttattat attccccaga
5941 acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca gccacttctt
6001 ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc cagctttcat
6061 ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc agacgtgcac
6121 tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc tgtacatcca
6181 caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc atagcatacc
6241 tctttttgac atacttcggg tatacatatc agtatatatt cttataccgc aaaaatcagc
6301 gcgcaaatac gcatactgtt atctggcttt tagtaagccc tctagaggat ccccgggtac
6361 cgagctcgaa ttccgatccc caattcctgg cagtttatgg cgggcgtcct gcccgccacc
6421 ctccgggccg ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc ctactcagga
6481 gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc
6541 gttttatttg atgcctgggg aattaattcc tggcagttta tggcggg
//
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