LOCUS       sequence-354667-        6406 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 6406)
  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 6406)
  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-KL (KanR, Lambda)
FEATURES             Location/Qualifiers
     source          1..6406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      115..201
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             433..738
                     /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     617..636
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     rep_origin      768..1356
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1257..1276
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    1416..1436
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1491..1518
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1567..1598)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     protein_bind    1675..1708
                     /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(1714..2427)
                     /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             2516..2524
                     /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             2669..3739
                     /codon_start=1
                     /gene="int"
                     /product="integrase from phage lambda"
                     /label=lambda integrase
                     /translation="MGRRRSHERRDLPPNLYIRNNGYYCYRDPRTGKEFGLGRDRRIAI
                     TEAIQANIELFSGHKHKPLTARINSDNSVTLHSWLDRYEKILASRGIKQKTLINYMSKI
                     KAIRRGLPDAPLEDITTKEIAAMLNGYIDEGKAASAKLIRSTLSDAFREAIAEGHITTN
                     HVAATRAAKSEVRRSRLTADEYLKIYQAAESSPCWLRLAMELAVVTGQRVGDLCEMKWS
                     DIVDGYLYVEQSKTGVKIAIPTALHIDALGISMKETLDKCKEILGGETIIASTRREPLS
                     SGTVSRYFMRARKASGLSFEGDPPTFHELRSLSARLYEKQISDKFAQHLLGHKSDTMAS
                     QYRDDRGREWDKIEIK"
     primer_bind     complement(3955..3974)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3966..4781
                     /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(4039..4058)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4903..5291
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    5302..5349
                     /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    5412..5645
                     /gene="attP"
                     /label=lambda attP
                     /bound_moiety="lambda integrase"
                     /note="integrase from phage lambda"
     terminator      complement(5769..6015)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(6106..6128)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6279..6297
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      join(6338..6406,1..18)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
       61 gggtggcggg caggacgccc gccataaact gccaggaatt aattccccag gcatcaaata
      121 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
      181 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
      241 ggagggtggc gggcaggacg cccgccataa actgccagga attggggatc ggaattcgag
      301 ctcggtaccc ggggatcctc tagagggctt actaaaagcc agataacagt atgcgtattt
      361 gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa gtatgtcaaa
      421 aagaggtatg ctatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg
      481 tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg
      541 gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc
      601 ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc
      661 ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg
      721 atgttctggg gaatataata acccgtagaa aagatcaaaa gatcttcttg agatcctttt
      781 tttctgcgcg taatctgcta cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
      841 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggctttag cagagcgcag
      901 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
      961 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1021 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1081 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1141 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     1201 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     1261 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     1321 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     1381 cggttcctgg ccttttgctg gccttttgct cacattacta gtagcggccg ctgcaggcat
     1441 gcctcgagat gcatggcgcc taacctaaac tgacaggcat caaattaagc agaaggccat
     1501 cctgacggat ggcctttttg cgtttcgaac aattgaaaaa acctcgcgcc ttacctgttg
     1561 agtaatagtc aaaagcctcc ggtcggaggc ttttgacttt ctgcttactg aatttcggtg
     1621 gtgccgttaa ttaaccggtg ggccctcatg ataataatgg tttcttagac gtccgaagtt
     1681 cctattctct agaaagtata ggaacttccc tagtcagcca aacgtctctt caggccactg
     1741 actagcgata actttcccca caacggaaca actctcattg catgggatca ttgggtactg
     1801 tgggtttagt ggttgtaaaa acacctgacc gctatccctg atcagtttct tgaaggtaaa
     1861 ctcatcaccc ccaagtctgg ctatgcagaa atcacctggc tcaacagcct gctcagggtc
     1921 aacgagaatt aacattccgt caggaaagct tggcttggag cctgttggtg cggtcatgga
     1981 attaccttca acctcaagcc agaatgcaga atcactggct tttttggttg tgcttaccca
     2041 tctctccgca tcacctttgg taaaggttct aagcttaggt gagaacatcc ctgcctgaac
     2101 atgagaaaaa acagggtact catactcact tctaagtgac ggctgcatac taaccgcttc
     2161 atacatctcg tagatttctc tggcgattga agggctaaat tcttcaacgc taactttgag
     2221 aatttttgta agcaatgcgg cgttataagc atttaatgca ttgatgccat taaataaagc
     2281 accaacgcct gactgcccca tccccatctt gtctgcgaca gattcctggg ataagccaag
     2341 ttcatttttc tttttttcat aaattgcttt aaggcgacgt gcgtcctcaa gctgctcttg
     2401 tgttaatggt ttcttttttg tgctcatacg ttaaatctat caccgcaagg gataaatatc
     2461 taacaccgtg cgtgttgact attttacctc tggcggtgat aatggttgca tgtactaagg
     2521 aggttgtata cgcgtcgtcg atgggttcgg gaatgcagga tattcccacc tccggttaag
     2581 gatggaagag agtatctgtt ccacgaatca gcggtaaagg ttgacttaaa tcgaccagta
     2641 acaggtggcc ttttgaagag gatcagaaat gggaagaagg cgaagtcatg agcgccggga
     2701 tttaccccct aacctttata taagaaacaa tggatattac tgctacaggg acccaaggac
     2761 gggtaaagag tttggattag gcagagacag gcgaatcgca atcactgaag ctatacaggc
     2821 caacattgag ttattttcag gacacaaaca caagcctctg acagcgagaa tcaacagtga
     2881 taattccgtt acgttacatt catggcttga tcgctacgaa aaaatcctgg ccagcagagg
     2941 aatcaagcag aagacactca taaattacat gagcaaaatt aaagcaataa ggaggggtct
     3001 gcctgatgct ccacttgaag acatcaccac aaaagaaatt gcggcaatgc tcaatggata
     3061 catagacgag ggcaaggcgg cgtcagccaa gttaatcaga tcaacactga gcgatgcatt
     3121 ccgagaggca atagctgaag gccatataac aacaaaccat gtcgctgcca ctcgcgcagc
     3181 aaaatcagag gtaaggagat caagacttac ggctgacgaa tacctgaaaa tttatcaagc
     3241 agcagaatca tcaccatgtt ggctcagact tgcaatggaa ctggctgttg ttaccgggca
     3301 acgagttggt gatttatgcg aaatgaagtg gtctgatatc gtagatggat atctttatgt
     3361 cgagcaaagc aaaacaggcg taaaaattgc catcccaaca gcattgcata ttgatgctct
     3421 cggaatatca atgaaggaaa cacttgataa atgcaaagag attcttggcg gagaaaccat
     3481 aattgcatct actcgtcgcg aaccgctttc atccggcaca gtatcaaggt attttatgcg
     3541 cgcacgaaaa gcatcaggtc tttccttcga aggggatccg cctacctttc acgagttgcg
     3601 cagtttgtct gcaagactct atgagaagca gataagcgat aagtttgctc aacatcttct
     3661 cgggcataag tcggacacca tggcatcaca gtatcgtgat gacagaggca gggagtggga
     3721 caaaattgaa atcaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa
     3781 attgataagc aatgccaagc ttggcactgg ctgatcagct agcccatggg tatggacagt
     3841 tttccctttg atatgtaacg cacgttgtgt ctcaaaatct ctgatgttac attgcacaag
     3901 ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     3961 gtgttatgag ccatattcaa cgggaaacgt cttgctcccg tccgcgctta aactccaaca
     4021 tggacgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     4081 caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     4141 gtagcgttgc caatgatgtt acagatgaga tggtccgtct caactggctg acggagttta
     4201 tgcctctccc gaccatcaag cattttatcc gtactcctga tgatgcgtgg ttactcacca
     4261 ccgcgattcc tgggaaaaca gccttccagg tattagaaga atatcctgat tcaggtgaaa
     4321 atattgttga tgcgctggcc gtgttcctgc gccggttaca ttcgattcct gtttgtaatt
     4381 gtccttttaa cagcgatcgt gtatttcgtc ttgctcaggc gcaatcacgc atgaataacg
     4441 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     4501 ggaaagaaat gcacaagctc ttgccattct caccggattc agtcgtcact catggtgatt
     4561 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     4621 gggtcggaat cgcagaccgt taccaggacc ttgccattct ttggaactgc ctcggtgagt
     4681 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     4741 ataaattgca gtttcatttg atgctcgatg agtttttcta ataatactag cagaaatcat
     4801 ccttagcgaa agctaaggat tttttttatc tgattaccgc ctttgagtga gcgtcgacct
     4861 agtgcggccg caagatccgg ccacgatgcg tccggcgtag aggatctgaa gatcagcagt
     4921 tcaacctgtt gatagtacgt actaagctct catgtttcac gtactaagct ctcatgttta
     4981 acgtactaag ctctcatgtt taacgaacta aaccctcatg gctaacgtac taagctctca
     5041 tggctaacgt actaagctct catgtttcac gtactaagct ctcatgtttg aacaataaaa
     5101 ttaatataaa tcagcaactt aaatagcctc taaggtttta agttttataa gaaaaaaaag
     5161 aatatataag gcttttaaag cttttaaggt ttaacggttg tggacaacaa gccagggatg
     5221 taacgcactg agaagccctt agagcctctc aaagcaattt tgagtgacac aggaacactt
     5281 aacggctgac atgggaatta ggaagttcct attccgaagt tcctattctc tagaaagtat
     5341 aggaacttcc atatgccatg gcatcacagt atcgtgatga cagaggcagg gagtgggaca
     5401 aaattgaaat caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat
     5461 tgataagcaa tgctttttta taatgccaac ttagtataaa aaagctgaac gagaaacgta
     5521 aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata
     5581 ctgtaaaaca caacatatat gcagtcacta tgaatcaact acttagatgg tattagtgac
     5641 ctgtaacaga gcattagcgc aaggtgattt ttgtcttctt gcgctaattt tttgtcatca
     5701 aacctgtcgc actccagaga agcacaaagc ctcgcaatcc agtgcaaagc tagcttcttc
     5761 gtctgtttct actggtattg gcacaaacct gattccaatt tgagcaaggc tatgtgccat
     5821 ctcgatactc gttcttaact caacagaaga tgctttgtgc atacagcccc tcgtttatta
     5881 tttatctcct cagccagccg ctgtgctttc agtggatttc ggataacaga aaggccggga
     5941 aatacccagc ctcgctttgt aacggagtag agacgaaagt gattgcgcct acccggatat
     6001 tatcgtgagg atgcgtcatc gccattaatt cactgatcag tgataagctg tcaaacatga
     6061 gaattgatcc ggctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     6121 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     6181 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     6241 tagcggagtg tatgctgcac atgacattaa cctataaaaa taggcgtatc acgaggccct
     6301 ttcgtcttca agaattaatt cccaattccc caggcatcaa ataaaacgaa aggctcagtc
     6361 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgct
//
