LOCUS       Exported                6769 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pOSIP-KT (KanR, P21)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6769)
  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 6769)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6769
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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      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             2299..3441
                     /codon_start=1
                     /gene="int"
                     /product="integrase from phage phi-21"
                     /label=phage phi-21 integrase
                     /translation="MAARPRSHKISIPNLYCKLDKRTGKVYWQYKHPLSGRFHSLGTDE
                     NEAKQVATEANTIIAEQRTRQILSVNERLERMKGRRSDITVTEWLDKYISIQEDRLQHN
                     ELRPNSYRQKGKPIRLFREHCGMQHLKDITALDIAEIIDAVKAEGHNRMAQVVRMVLID
                     VFKEAQHAGHVPPGFNPAQATKQPRNRVNRQRLSLLEWQAIFESVSRRQPYLKCGMLLA
                     LVTGQRLGDICNLKFSDIWDDMLHITQEKTGSKLAIPLNLKCDALNITLREVISQCRDA
                     VVSKYLVHYRHTTSQANRGDQVSANTLTTAFKKAKEKCGIKWEQGTAPTFHEQRSLSER
                     LYREQGLDTQKLLGHKSRKMTDRYNDDRGKDWIIVDIKTA"
     primer_bind     complement(3665..3684)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3676..4491
                     /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(3749..3768)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4613..5001
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    5012..5059
                     /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    5139..5638
                     /label=phage phi-21 attP
                     /bound_moiety="phage phi-21 integrase"
                     /note="attachment site of phage phi-21"
     terminator      5659..5905
                     /label=-lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(5995..6017)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6168..6186
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      6227..6313
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6410..6496
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6593..6679
                     /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 cgcgtagaac cgcaactccc aataaacgca aacccaaaac
     2281 tccaacggat aatcgctgat ggctgctaga ccccgatctc acaaaatctc tatacccaat
     2341 ttatattgca aattagataa gcgaaccgga aaggtatatt ggcaatacaa acatccacta
     2401 tccggtcgtt ttcatagctt aggaactgat gagaatgaag caaaacaagt tgctactgaa
     2461 gcaaatacca ttattgctga acaacgtacc agacaaatat taagcgtcaa tgagcgtctg
     2521 gaaagaatga aaggcaggcg ctcagacatt acggtgacag aatggcttga taaatatatt
     2581 tctatccagg aggacaggct gcaacataat gaactaagac ccaactccta tcggcaaaaa
     2641 ggcaaaccca ttcgtctttt ccgtgagcat tgtggaatgc aacacctcaa ggatattacc
     2701 gcacttgata ttgccgaaat aattgatgct gtaaaggctg aaggtcataa caggatggcg
     2761 caagtcgtga gaatggtgtt gatcgacgtc ttcaaagaag cacaacacgc aggacatgtt
     2821 ccgccaggat ttaacccagc gcaggcaaca aaacaaccgc gaaatcgagt aaaccgccaa
     2881 agattgtcac tgctcgaatg gcaggcaata tttgaaagcg taagcagacg gcagccctat
     2941 ttaaaatgcg gcatgctact tgctcttgtt actggacaac gtttaggcga tatctgcaat
     3001 ttgaaattct ctgatatatg ggacgacatg ttgcacatta ctcaggaaaa aaccggttca
     3061 aaacttgcta ttccgcttaa cctgaaatgc gatgctctga atattaccct tcgtgaagtt
     3121 atatctcagt gcagggatgc tgttgttagt aaatatctgg tccattaccg tcacactacc
     3181 tctcaagcaa acagaggaga ccaggtttct gcaaatactc tgacaacggc ttttaaaaag
     3241 gccaaggaaa aatgtggcat aaaatgggag caaggaactg cgcccacatt tcatgaacag
     3301 cgatctctgt cagaacgatt atatcgggaa cagggtctgg atacgcaaaa gttgttaggc
     3361 cataaatcca gaaaaatgac cgaccgatac aatgatgatc gtggtaaaga ctggattatc
     3421 gtagatatca aaacagcata gaaaatagcc agttttgggg aagggttttg gggaaagttt
     3481 tggggaagat tttacatcat cataaaacaa cgggcgtgtt atacgcccgt tatccatggg
     3541 tatggacagt tttccctttg atatgtaacg cacgttgtgt ctcaaaatct ctgatgttac
     3601 attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt
     3661 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctcccg tccgcgctta
     3721 aactccaaca tggacgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     3781 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa
     3841 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtccgtct caactggctg
     3901 acggagttta tgcctctccc gaccatcaag cattttatcc gtactcctga tgatgcgtgg
     3961 ttactcacca ccgcgattcc tgggaaaaca gccttccagg tattagaaga atatcctgat
     4021 tcaggtgaaa atattgttga tgcgctggcc gtgttcctgc gccggttaca ttcgattcct
     4081 gtttgtaatt gtccttttaa cagcgatcgt gtatttcgtc ttgctcaggc gcaatcacgc
     4141 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     4201 gaacaagtct ggaaagaaat gcacaagctc ttgccattct caccggattc agtcgtcact
     4261 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     4321 gatgttggac gggtcggaat cgcagaccgt taccaggacc ttgccattct ttggaactgc
     4381 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     4441 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta ataatactag
     4501 cagaaatcat ccttagcgaa agctaaggat tttttttatc tgattaccgc ctttgagtga
     4561 gcgtcgacct agtgcggccg caagatccgg ccacgatgcg tccggcgtag aggatctgaa
     4621 gatcagcagt tcaacctgtt gatagtacgt actaagctct catgtttcac gtactaagct
     4681 ctcatgttta acgtactaag ctctcatgtt taacgaacta aaccctcatg gctaacgtac
     4741 taagctctca tggctaacgt actaagctct catgtttcac gtactaagct ctcatgtttg
     4801 aacaataaaa ttaatataaa tcagcaactt aaatagcctc taaggtttta agttttataa
     4861 gaaaaaaaag aatatataag gcttttaaag cttttaaggt ttaacggttg tggacaacaa
     4921 gccagggatg taacgcactg agaagccctt agagcctctc aaagcaattt tgagtgacac
     4981 aggaacactt aacggctgac atgggaatta ggaagttcct attccgaagt tcctattctc
     5041 tagaaagtat aggaacttcc atatgccatg gaatgaccga ccgatacaat gatgatcgtg
     5101 gtaaagactg gattatcgta gatatcaaaa cagcatagaa aatagccagt tttggggaag
     5161 ggttttgggg aaagttttgg ggaagatttt acatcatcat aaaacaacgg gcgtgttata
     5221 cgcccgttat aatatttaac acatgtggta attacatatt tttgatgatc gcgtcaccaa
     5281 actcgctgca tttcagcagt ttagcgcctt ccatcagacg ttcgaagtca taggttacgg
     5341 tcttggcgtt gattgcgcct tccatacctt taacaatcag gtctgcggct tcagtccagc
     5401 ccatatggcg cagcagtata taaaaattat gtattcaact cgctgatatt taatcataaa
     5461 tattattttc ttgtctttgt attagccaaa atcaacttta ttcaaccgtt tgatttcatt
     5521 gacgttcgtt tttattttgg ggaaagattt tctctaccat ttatatcaaa attacaagtc
     5581 tacattttat taacatctca atacatgtgt ggccttgcaa gccattgcga ggccttatgc
     5641 tagcttcttc gtctgtttct actggtattg gcacaaacct gattccaatt tgagcaaggc
     5701 tatgtgccat ctcgatactc gttcttaact caacagaaga tgctttgtgc atacagcccc
     5761 tcgtttatta tttatctcct cagccagccg ctgtgctttc agtggatttc ggataacaga
     5821 aaggccggga aatacccagc ctcgctttgt aacggagtag agacgaaagt gattgcgcct
     5881 acccggatat tatcgtgagg atgcgtcatc gccattaatt cactgatcag tgatagctgt
     5941 caaacatgag aattgatccg gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     6001 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     6061 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     6121 acgtagcgat agcggagtgt atgctgcaca tgacattaac ctataaaaat aggcgtatca
     6181 cgaggccctt tcgtcttcaa gaattaattc ccaattcccc aggcatcaaa taaaacgaaa
     6241 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     6301 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     6361 gcgggcagga cgcccgccat aaactgccag gaattaattc cccaggcatc aaataaaacg
     6421 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     6481 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg
     6541 gtggcgggca ggacgcccgc cataaactgc caggaattaa ttccccaggc atcaaataaa
     6601 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     6661 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg
     6721 agggtggcgg gcaggacgcc cgccataaac tgccaggaat tggggatcg
//
