LOCUS       sequence-64643-e        6299 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 6299)
  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 6299)
  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-KH (KanR, HK022)
FEATURES             Location/Qualifiers
     source          1..6299
                     /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             2270..3343
                     /codon_start=1
                     /gene="int"
                     /product="integrase from phage HK022"
                     /label=HK022 integrase
                     /translation="MGRRRSHERRDLPPNLYIRNNGYYCYRDPRTGKEFGLGRDRRIAI
                     TEAIQANIELLSGNRRESLIDRIKGADAITLHAWLDRYETILSERGIRPKTLLDYASKI
                     RAIRRKLPDKPLADISTKEVAAMLNTYVAEGKSASAKLIRSTLVDVFREAIAEGHVATN
                     PVTATRTAKSEVRRSRLTANEYVAIYHAAEPLPIWLRLAMDLAVVTGQRVGDLCRMKWS
                     DINDNHLHIEQSKTGAKLAIPLTLTIDALNISLADTLQQCREASSSETIIASKHHDPLS
                     PKTVSKYFTKARNASGLSFDGNPPTFHELRSLSARLYRNQIGDKFAQRLLGHKSDSMAA
                     RYRDSRGREWDKIEIDK"
     primer_bind     complement(3504..3523)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3515..4330
                     /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(3588..3607)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4452..4840
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    4851..4898
                     /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    4928..5154
                     /gene="attP"
                     /label=HK022 attP
                     /bound_moiety="HK022 integrase"
                     /note="attachment site of phage HK022"
     terminator      complement(5189..5435)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(5525..5547)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5698..5716
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      5757..5843
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5940..6026
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6123..6209
                     /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 cgcgtgtagc cttttgaaga ggatcagaaa tgggaagaag
     2281 gcgaagtcat gagcgccggg atttaccccc taacctttat ataagaaaca atggatatta
     2341 ctgctacagg gacccaagga cgggtaaaga gtttggatta ggccgagaca ggcgaatcgc
     2401 aatcactgaa gctatacagg ccaacattga gttgctatcc gggaacaggc gtgagtcact
     2461 gatagacaga attaaaggcg ctgacgcaat cactcttcat gcgtggcttg accgatatga
     2521 aacaatcctc agcgagaggg gtatcaggcc gaaaactcta ctcgactacg ccagcaaaat
     2581 cagagcaatc cgaagaaaat tgccggacaa accgctcgct gacatatcaa cgaaagaggt
     2641 ggcagcaatg ctaaacacct acgtagcaga aggcaaatcg gcttccgcaa aattgatcag
     2701 gtcaaccctt gttgacgttt ttcgtgaggc aatagccgag gggcatgtgg ctacgaatcc
     2761 ggtaacagca acccgcacag caaaatcaga agtaaggcgt tcaagactga cagctaatga
     2821 atatgtcgcg atataccatg cagccgaacc gctcccaatc tggctgagac tggcaatgga
     2881 tttggctgtc gttacagggc agagagtagg cgatttgtgc agaatgaaat ggtcagacat
     2941 aaacgacaac catcttcaca ttgaacagag taaaacaggg gctaaactcg ccattccact
     3001 aacgctaacg attgacgcgc tcaatatctc attggctgat acactacagc aatgcaggga
     3061 ggccagcagc agtgaaacta taatcgcatc aaagcatcac gatccgcttt ccccgaaaac
     3121 agtatcaaag tattttacaa aggcgagaaa tgcatctgga ctctcatttg atggaaaccc
     3181 gccaacattc catgaactgc gtagcctgtc agcgaggcta taccggaacc agattggcga
     3241 taagtttgct caacgtcttc tcgggcataa atcagattca atggcggcgc ggtataggga
     3301 cagccgtgga cgggaatggg acaaaattga aatcgacaaa tgattttatt ttgactaata
     3361 atgacctact taccatgggt atggacagtt ttccctttga tatgtaacgc acgttgtgtc
     3421 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
     3481 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     3541 ttgctcccgt ccgcgcttaa actccaacat ggacgctgat ttatatgggt ataaatgggc
     3601 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     3661 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     3721 ggtccgtctc aactggctga cggagtttat gcctctcccg accatcaagc attttatccg
     3781 tactcctgat gatgcgtggt tactcaccac cgcgattcct gggaaaacag ccttccaggt
     3841 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggccg tgttcctgcg
     3901 ccggttacat tcgattcctg tttgtaattg tccttttaac agcgatcgtg tatttcgtct
     3961 tgctcaggcg caatcacgca tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     4021 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cacaagctct tgccattctc
     4081 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     4141 gaaattaata ggttgtattg atgttggacg ggtcggaatc gcagaccgtt accaggacct
     4201 tgccattctt tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     4261 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     4321 gtttttctaa taatactagc agaaatcatc cttagcgaaa gctaaggatt ttttttatct
     4381 gattaccgcc tttgagtgag cgtcgaccta gtgcggccgc aagatccggc cacgatgcgt
     4441 ccggcgtaga ggatctgaag atcagcagtt caacctgttg atagtacgta ctaagctctc
     4501 atgtttcacg tactaagctc tcatgtttaa cgtactaagc tctcatgttt aacgaactaa
     4561 accctcatgg ctaacgtact aagctctcat ggctaacgta ctaagctctc atgtttcacg
     4621 tactaagctc tcatgtttga acaataaaat taatataaat cagcaactta aatagcctct
     4681 aaggttttaa gttttataag aaaaaaaaga atatataagg cttttaaagc ttttaaggtt
     4741 taacggttgt ggacaacaag ccagggatgt aacgcactga gaagccctta gagcctctca
     4801 aagcaatttt gagtgacaca ggaacactta acggctgaca tgggaattag gaagttccta
     4861 ttccgaagtt cctattctct agaaagtata ggaacttcca tatgccatgg gacaaaattg
     4921 aaatcgacaa atgattttat tttgactaat aatgacctac ttacattaat ttactgataa
     4981 ttaaagagat tttaaatata caacttattc acctaaagga tgacaaaata acattaatca
     5041 cttaaaaatc atcgcattac actaatctgt ggttaaatga tagactacat aatgcgacaa
     5101 aacgcaacat atccagtcac tatgaatcaa ctacttagat agtattagtg acctgagaca
     5161 gagcattagc tagcttcttc gtctgtttct actggtattg gcacaaacct gattccaatt
     5221 tgagcaaggc tatgtgccat ctcgatactc gttcttaact caacagaaga tgctttgtgc
     5281 atacagcccc tcgtttatta tttatctcct cagccagccg ctgtgctttc agtggatttc
     5341 ggataacaga aaggccggga aatacccagc ctcgctttgt aacggagtag agacgaaagt
     5401 gattgcgcct acccggatat tatcgtgagg atgcgtcatc gccattaatt cactgatcag
     5461 tgatagctgt caaacatgag aattgatccg gctgcctcgc gcgtttcggt gatgacggtg
     5521 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     5581 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     5641 tgacccagtc acgtagcgat agcggagtgt atgctgcaca tgacattaac ctataaaaat
     5701 aggcgtatca cgaggccctt tcgtcttcaa gaattaattc ccaattcccc aggcatcaaa
     5761 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     5821 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc
     5881 ccggagggtg gcgggcagga cgcccgccat aaactgccag gaattaattc cccaggcatc
     5941 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     6001 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     6061 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggaattaa ttccccaggc
     6121 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     6181 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc
     6241 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggaat tggggatcg
//
