LOCUS       Exported                6763 bp ds-DNA     circular SYN 16-JUN-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pOSIP-KP (KanR, Phi80)
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 Jun 16, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6763
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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      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      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
//
