LOCUS       sequence-352814-        6971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6971)
  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 6971)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.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-KC (KanR, PhiC31)
FEATURES             Location/Qualifiers
     source          1..6971
                     /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             2272..4089
                     /codon_start=1
                     /product="integrase from phage phi-C31"
                     /label=phage phi-C31 integrase
                     /translation="MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDG
                     GRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIP
                     IVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQREL
                     GGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWRE
                     IKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRIL
                     RDPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQA
                     WLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPG
                     QHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERA
                     NLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAE
                     APKLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPI
                     EKRASITWAKPPTDDDEDDAQDGTEDVAA"
     primer_bind     complement(4213..4232)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4224..5039
                     /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(4297..4316)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5161..5549
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    5560..5607
                     /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    5676..5775
                     /label=phage phi-C31 attP
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attachment site of phage phi-C31"
     terminator      complement(5860..6106)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(6197..6219)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6370..6388
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      6429..6515
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6612..6698
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6795..6881
                     /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 aggttgtatc ctgcaggccg agaaaaagga ggagataaac agtggacacg
     2281 tacgcgggtg cttacgaccg tcagtcgcgc gagcgcgaaa attcgagcgc agcaagccca
     2341 gcgacacagc gtagcgccaa cgaagacaag gcggccgacc ttcagcgcga agtcgagcgc
     2401 gacgggggcc ggttcaggtt cgtcgggcat ttcagcgaag cgccgggcac gtcggcgttc
     2461 gggacggcgg agcgcccgga gttcgaacgc atcctgaacg aatgccgcgc cgggcggctc
     2521 aacatgatca ttgtctatga cgtgtcgcgc ttctcgcgcc tgaaggtcat ggacgcgatt
     2581 ccgattgtct cggaattgct cgccctgggc gtgacgattg tttccactca ggaaggcgtc
     2641 ttccggcagg gaaacgtcat ggacctgatt cacctgatta tgcggctcga cgcgtcgcac
     2701 aaagaatctt cgctgaagtc ggcgaagatt ctcgacacga agaaccttca gcgcgaattg
     2761 ggcgggtacg tcggcgggaa ggcgccttac ggcttcgagc ttgtttcgga gacgaaggag
     2821 atcacgcgca acggccgaat ggtcaatgtc gtcatcaaca agcttgcgca ctcgaccact
     2881 ccccttaccg gacccttcga gttcgagccc gacgtaatcc ggtggtggtg gcgtgagatc
     2941 aagacgcaca aacaccttcc cttcaagccg ggcagtcaag ccgccattca cccgggcagc
     3001 atcacggggc tttgtaagcg catggacgct gacgccgtgc cgacccgggg cgagacgatt
     3061 gggaagaaga ccgcttcaag cgcctgggac ccggcaaccg ttatgcgaat ccttcgggac
     3121 ccgcgtattg cgggcttcgc cgctgaggtg atctacaaga agaagccgga cggcacgccg
     3181 accacgaaga ttgagggtta ccgcattcag cgcgacccga tcacgctccg gccggtcgag
     3241 cttgattgcg gaccgatcat cgagcccgct gagtggtatg agcttcaggc gtggttggac
     3301 ggcagggggc gcggcaaggg gctttcccgg gggcaagcca ttctgtccgc catggacaag
     3361 ctgtactgcg agtgtggcgc cgtcatgact tcgaagcgcg gggaagaatc gatcaaggac
     3421 tcttaccgct gccgtcgccg gaaggtggtc gacccgtccg cacctgggca gcacgaaggc
     3481 acgtgcaacg tcagcatggc ggcactcgac aagttcgttg cggaacgcat cttcaacaag
     3541 atcaggcacg ccgaaggcga cgaagagacg ttggcgcttc tgtgggaagc cgcccgacgc
     3601 ttcggcaagc tcactgaggc gcctgagaag agcggcgaac gggcgaacct tgttgcggag
     3661 cgcgccgacg ccctgaacgc ccttgaagag ctgtacgaag accgcgcggc aggcgcgtac
     3721 gacggacccg ttggcaggaa gcacttccgg aagcaacagg cagcgctgac gctccggcag
     3781 caaggggcgg aagagcggct tgccgaactt gaagccgccg aagccccgaa gcttcccctt
     3841 gaccaatggt tccccgaaga cgccgacgct gacccgaccg gccctaagtc gtggtggggg
     3901 cgcgcgtcag tagacgacaa gcgcgtgttc gtcgggctct tcgtagacaa gatcgttgtc
     3961 acgaagtcga ctacgggcag ggggcaggga acgcccatcg agaagcgcgc ttcgatcacg
     4021 tgggcgaagc cgccgaccga cgacgacgaa gacgacgccc aggacggcac ggaagacgta
     4081 gcggcgtagt tttattttga ctaataatga cctacttaca cgttgtgtct caaaatctct
     4141 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
     4201 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctcccgtc
     4261 cgcgcttaaa ctccaacatg gacgctgatt tatatgggta taaatgggct cgcgataatg
     4321 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
     4381 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtccgtctca
     4441 actggctgac ggagtttatg cctctcccga ccatcaagca ttttatccgt actcctgatg
     4501 atgcgtggtt actcaccacc gcgattcctg ggaaaacagc cttccaggta ttagaagaat
     4561 atcctgattc aggtgaaaat attgttgatg cgctggccgt gttcctgcgc cggttacatt
     4621 cgattcctgt ttgtaattgt ccttttaaca gcgatcgtgt atttcgtctt gctcaggcgc
     4681 aatcacgcat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     4741 ggcctgttga acaagtctgg aaagaaatgc acaagctctt gccattctca ccggattcag
     4801 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     4861 gttgtattga tgttggacgg gtcggaatcg cagaccgtta ccaggacctt gccattcttt
     4921 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     4981 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     5041 aatactagca gaaatcatcc ttagcgaaag ctaaggattt tttttatctg attaccgcct
     5101 ttgagtgagc gtcgacctag tgcggccgca agatccggcc acgatgcgtc cggcgtagag
     5161 gatctgaaga tcagcagttc aacctgttga tagtacgtac taagctctca tgtttcacgt
     5221 actaagctct catgtttaac gtactaagct ctcatgttta acgaactaaa ccctcatggc
     5281 taacgtacta agctctcatg gctaacgtac taagctctca tgtttcacgt actaagctct
     5341 catgtttgaa caataaaatt aatataaatc agcaacttaa atagcctcta aggttttaag
     5401 ttttataaga aaaaaaagaa tatataaggc ttttaaagct tttaaggttt aacggttgtg
     5461 gacaacaagc cagggatgta acgcactgag aagcccttag agcctctcaa agcaattttg
     5521 agtgacacag gaacacttaa cggctgacat gggaattagg aagttcctat tccgaagttc
     5581 ctattctcta gaaagtatag gaacttccat atgccatggt cgcgctcgcg cgactgacgg
     5641 tcgtaagcac ccgcgtacgt gtccaccccg gtcacaaccc cttgtgtcat gtcggcggcc
     5701 ctacgccccc aactgagaga actcaaaggt taccccagtt ggggcactac tcccgaaaac
     5761 cgcttctgac ctgggaaaac gtgaagcccc ggggcatccg ctgagggttg ccgccggggc
     5821 ttcggtgtgt ccgtcagtag ctagcttctt cgtctgtttc tactggtatt ggcacaaacc
     5881 tgattccaat ttgagcaagg ctatgtgcca tctcgatact cgttcttaac tcaacagaag
     5941 atgctttgtg catacagccc ctcgtttatt atttatctcc tcagccagcc gctgtgcttt
     6001 cagtggattt cggataacag aaaggccggg aaatacccag cctcgctttg taacggagta
     6061 gagacgaaag tgattgcgcc tacccggata ttatcgtgag gatgcgtcat cgccattaat
     6121 tcactgatca gtgataagct gtcaaacatg agaattgatc cggctgcctc gcgcgtttcg
     6181 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     6241 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     6301 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatgctgca catgacatta
     6361 acctataaaa ataggcgtat cacgaggccc tttcgtcttc aagaattaat tcccaattcc
     6421 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     6481 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     6541 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggaattaat
     6601 tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     6661 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     6721 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggaatt
     6781 aattccccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     6841 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     6901 acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccagga
     6961 attggggatc g
//
