LOCUS       Exported                6587 bp ds-DNA     circular SYN 16-FEB-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pOSIP-KT (KanR, P21)
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 Feb 16, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6587
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      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      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      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
//
