LOCUS pOSIP-KH (KanR, HK022) 6300 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 6300)
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 6300)
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-KH (KanR, HK022)
FEATURES Location/Qualifiers
source 1..6300
/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 2270..3343
/codon_start=1
/gene="int"
/product="integrase from phage HK022"
/label=HK022 integrase
/translation="MGRRRSHERRDLPPNLYIRNNGYYCYRDPRTGKEFGLGRDRRIAI
TEAIQANIELLSGNRRESLIDRIKGADAITLHAWLDRYETILSERGIRPKTLLDYASKI
RAIRRKLPDKPLADISTKEVAAMLNTYVAEGKSASAKLIRSTLVDVFREAIAEGHVATN
PVTATRTAKSEVRRSRLTANEYVAIYHAAEPLPIWLRLAMDLAVVTGQRVGDLCRMKWS
DINDNHLHIEQSKTGAKLAIPLTLTIDALNISLADTLQQCREASSSETIIASKHHDPLS
PKTVSKYFTKARNASGLSFDGNPPTFHELRSLSARLYRNQIGDKFAQRLLGHKSDSMAA
RYRDSRGREWDKIEIDK"
primer_bind complement(3504..3523)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 3515..4330
/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(3588..3607)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
rep_origin 4452..4840
/label=R6K gamma ori
/note="gamma replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication"
protein_bind 4851..4898
/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 4928..5154
/gene="attP"
/label=HK022 attP
/bound_moiety="HK022 integrase"
/note="attachment site of phage HK022"
terminator complement(5189..5435)
/label=lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
primer_bind complement(5526..5548)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 5699..5717
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
terminator 5758..5844
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5941..6027
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 6124..6210
/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 aggttgtata cgcgtgtagc cttttgaaga ggatcagaaa tgggaagaag
2281 gcgaagtcat gagcgccggg atttaccccc taacctttat ataagaaaca atggatatta
2341 ctgctacagg gacccaagga cgggtaaaga gtttggatta ggccgagaca ggcgaatcgc
2401 aatcactgaa gctatacagg ccaacattga gttgctatcc gggaacaggc gtgagtcact
2461 gatagacaga attaaaggcg ctgacgcaat cactcttcat gcgtggcttg accgatatga
2521 aacaatcctc agcgagaggg gtatcaggcc gaaaactcta ctcgactacg ccagcaaaat
2581 cagagcaatc cgaagaaaat tgccggacaa accgctcgct gacatatcaa cgaaagaggt
2641 ggcagcaatg ctaaacacct acgtagcaga aggcaaatcg gcttccgcaa aattgatcag
2701 gtcaaccctt gttgacgttt ttcgtgaggc aatagccgag gggcatgtgg ctacgaatcc
2761 ggtaacagca acccgcacag caaaatcaga agtaaggcgt tcaagactga cagctaatga
2821 atatgtcgcg atataccatg cagccgaacc gctcccaatc tggctgagac tggcaatgga
2881 tttggctgtc gttacagggc agagagtagg cgatttgtgc agaatgaaat ggtcagacat
2941 aaacgacaac catcttcaca ttgaacagag taaaacaggg gctaaactcg ccattccact
3001 aacgctaacg attgacgcgc tcaatatctc attggctgat acactacagc aatgcaggga
3061 ggccagcagc agtgaaacta taatcgcatc aaagcatcac gatccgcttt ccccgaaaac
3121 agtatcaaag tattttacaa aggcgagaaa tgcatctgga ctctcatttg atggaaaccc
3181 gccaacattc catgaactgc gtagcctgtc agcgaggcta taccggaacc agattggcga
3241 taagtttgct caacgtcttc tcgggcataa atcagattca atggcggcgc ggtataggga
3301 cagccgtgga cgggaatggg acaaaattga aatcgacaaa tgattttatt ttgactaata
3361 atgacctact taccatgggt atggacagtt ttccctttga tatgtaacgc acgttgtgtc
3421 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
3481 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
3541 ttgctcccgt ccgcgcttaa actccaacat ggacgctgat ttatatgggt ataaatgggc
3601 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
3661 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
3721 ggtccgtctc aactggctga cggagtttat gcctctcccg accatcaagc attttatccg
3781 tactcctgat gatgcgtggt tactcaccac cgcgattcct gggaaaacag ccttccaggt
3841 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggccg tgttcctgcg
3901 ccggttacat tcgattcctg tttgtaattg tccttttaac agcgatcgtg tatttcgtct
3961 tgctcaggcg caatcacgca tgaataacgg tttggttgat gcgagtgatt ttgatgacga
4021 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cacaagctct tgccattctc
4081 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
4141 gaaattaata ggttgtattg atgttggacg ggtcggaatc gcagaccgtt accaggacct
4201 tgccattctt tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
4261 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
4321 gtttttctaa taatactagc agaaatcatc cttagcgaaa gctaaggatt ttttttatct
4381 gattaccgcc tttgagtgag cgtcgaccta gtgcggccgc aagatccggc cacgatgcgt
4441 ccggcgtaga ggatctgaag atcagcagtt caacctgttg atagtacgta ctaagctctc
4501 atgtttcacg tactaagctc tcatgtttaa cgtactaagc tctcatgttt aacgaactaa
4561 accctcatgg ctaacgtact aagctctcat ggctaacgta ctaagctctc atgtttcacg
4621 tactaagctc tcatgtttga acaataaaat taatataaat cagcaactta aatagcctct
4681 aaggttttaa gttttataag aaaaaaaaga atatataagg cttttaaagc ttttaaggtt
4741 taacggttgt ggacaacaag ccagggatgt aacgcactga gaagccctta gagcctctca
4801 aagcaatttt gagtgacaca ggaacactta acggctgaca tgggaattag gaagttccta
4861 ttccgaagtt cctattctct agaaagtata ggaacttcca tatgccatgg gacaaaattg
4921 aaatcgacaa atgattttat tttgactaat aatgacctac ttacattaat ttactgataa
4981 ttaaagagat tttaaatata caacttattc acctaaagga tgacaaaata acattaatca
5041 cttaaaaatc atcgcattac actaatctgt ggttaaatga tagactacat aatgcgacaa
5101 aacgcaacat atccagtcac tatgaatcaa ctacttagat agtattagtg acctgagaca
5161 gagcattagc tagcttcttc gtctgtttct actggtattg gcacaaacct gattccaatt
5221 tgagcaaggc tatgtgccat ctcgatactc gttcttaact caacagaaga tgctttgtgc
5281 atacagcccc tcgtttatta tttatctcct cagccagccg ctgtgctttc agtggatttc
5341 ggataacaga aaggccggga aatacccagc ctcgctttgt aacggagtag agacgaaagt
5401 gattgcgcct acccggatat tatcgtgagg atgcgtcatc gccattaatt cactgatcag
5461 tgataagctg tcaaacatga gaattgatcc ggctgcctcg cgcgtttcgg tgatgacggt
5521 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
5581 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
5641 atgacccagt cacgtagcga tagcggagtg tatgctgcac atgacattaa cctataaaaa
5701 taggcgtatc acgaggccct ttcgtcttca agaattaatt cccaattccc caggcatcaa
5761 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
5821 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
5881 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggaattaatt ccccaggcat
5941 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
6001 gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa
6061 cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggaatta attccccagg
6121 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
6181 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
6241 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggaa ttggggatcg
//
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