LOCUS       sequence-352787-        6751 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 6751)
  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 6751)
  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-TH (TetR, HK022)
FEATURES             Location/Qualifiers
     source          1..6751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(83..169)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(210..228)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     379..401
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     terminator      492..738
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     protein_bind    773..999
                     /gene="attP"
                     /label=HK022 attP
                     /bound_moiety="HK022 integrase"
                     /note="attachment site of phage HK022"
     protein_bind    complement(1029..1076)
                     /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      1087..1475
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(1532..1626)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1685..2890)
                     /codon_start=1
                     /gene="tetA from transposon Tn10"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MNSSTKIALVITLLDAMGIGLIMPVLPTLLREFIASEDIANHFGV
                     LLALYALMQVIFAPWLGKMSDRFGRRPVLLLSLIGASLDYLLLAFSSALWMLYLGRLLS
                     GITGATGAVAASVIADTTSASQRVKWFGWLGASFGLGLIAGPIIGGFAGEISPHSPFFI
                     AALLNIVTFLVVMFWFRETKNTRDNTDTEVGVETQSNSVYITLFKTMPILLIIYFSAQL
                     IGQIPATVWVLFTENRFGWNSMMVGFSLAGLGLLHSVFQAFVAGRIATKWGEKTAVLLG
                     FIADSSAFAFLAFISEGWLVFPVLILLAGGGIALPALQGVMSIQTKSHQQGALQGLLVS
                     LTNATGVIGPLLFAVIYNHSLPIWDGWIWIIGLAFYCIIILLSMTFMLTPQAQGSKQET
                     SA"
     promoter        2898..2952
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    2899..2917
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             complement(3035..4108)
                     /codon_start=1
                     /gene="int"
                     /product="integrase from phage HK022"
                     /label=HK022 integrase
                     /translation="MGRRRSHERRDLPPNLYIRNNGYYCYRDPRTGKEFGLGRDRRIAI
                     TEAIQANIELLSGNRRESLIDRIKGADAITLHAWLDRYETILSERGIRPKTLLDYASKI
                     RAIRRKLPDKPLADISTKEVAAMLNTYVAEGKSASAKLIRSTLVDVFREAIAEGHVATN
                     PVTATRTAKSEVRRSRLTANEYVAIYHAAEPLPIWLRLAMDLAVVTGQRVGDLCRMKWS
                     DINDNHLHIEQSKTGAKLAIPLTLTIDALNISLADTLQQCREASSSETIIASKHHDPLS
                     PKTVSKYFTKARNASGLSFDGNPPTFHELRSLSARLYRNQIGDKFAQRLLGHKSDSMAA
                     RYRDSRGREWDKIEIDK"
     RBS             4144..4152
                     /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             4241..4954
                     /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(4961..4994)
                     /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      5071..5102
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     terminator      complement(5151..5178)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     misc_feature    5233..5253
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     rep_origin      complement(5313..5901)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5393..5412)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5931..6236)
                     /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(6033..6052)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      complement(6468..6554)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(6651..6737)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aattcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcgcaa
       61 cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac
      121 agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctgggga
      181 attgggaatt aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
      241 tgtcatgtgc agcatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
      301 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
      361 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
      421 aacgcgcgag gcagccggat caattctcat gtttgacagc ttatcactga tcagtgaatt
      481 aatggcgatg acgcatcctc acgataatat ccgggtaggc gcaatcactt tcgtctctac
      541 tccgttacaa agcgaggctg ggtatttccc ggcctttctg ttatccgaaa tccactgaaa
      601 gcacagcggc tggctgagga gataaataat aaacgagggg ctgtatgcac aaagcatctt
      661 ctgttgagtt aagaacgagt atcgagatgg cacatagcct tgctcaaatt ggaatcaggt
      721 ttgtgccaat accagtagaa acagacgaag aagctagcta atgctctgtc tcaggtcact
      781 aatactatct aagtagttga ttcatagtga ctggatatgt tgcgttttgt cgcattatgt
      841 agtctatcat ttaaccacag attagtgtaa tgcgatgatt tttaagtgat taatgttatt
      901 ttgtcatcct ttaggtgaat aagttgtata tttaaaatct ctttaattat cagtaaatta
      961 atgtaagtag gtcattatta gtcaaaataa aatcatttgt cgatttcaat tttgtcccat
     1021 ggcatatgga agttcctata ctttctagag aataggaact tcggaatagg aacttcctaa
     1081 ttcccatgtc agccgttaag tgttcctgtg tcactcaaaa ttgctttgag aggctctaag
     1141 ggcttctcag tgcgttacat ccctggcttg ttgtccacaa ccgttaaacc ttaaaagctt
     1201 taaaagcctt atatattctt ttttttctta taaaacttaa aaccttagag gctatttaag
     1261 ttgctgattt atattaattt tattgttcaa acatgagagc ttagtacgtg aaacatgaga
     1321 gcttagtacg ttagccatga gagcttagta cgttagccat gagggtttag ttcgttaaac
     1381 atgagagctt agtacgttaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg
     1441 tactatcaac aggttgaact gctgatcttc agatcctcta cgccggacgc atcgtggccg
     1501 gatcttgcgg ccgcactagg tcgacgcttg gattctcacc aataaaaaac gcccggcggc
     1561 aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca
     1621 ggagtccaag cgagctcaaa aattaaaaat gaagttttgg aggcctcatt tggtgacgaa
     1681 ataactaagc acttgtctcc tgtttactcc cctgagcttg aggggtcaac atgaaggtca
     1741 ttgatagcag gataataata cagtaaaacg ctaaaccaat aatccaaatc cagccatccc
     1801 aaattggtag tgaatgatta taaataacag caaacagtaa tgggccaata acaccggttg
     1861 cattggtaag gctcaccaat aatccctgta aagcaccttg ctgatgactc tttgtttgga
     1921 tagacatcac tccctgtaat gcaggtaaag cgatcccacc accagccaat aaaattaaaa
     1981 cagggaaaac taaccaacct tcagatataa acgctaaaaa ggcaaatgca ctactatctg
     2041 caataaatcc gagcagtact gccgtttttt cgccccattt agtggctatt cttcctgcca
     2101 caaaggcttg gaatactgag tgtaaaagac caagacccgc taatgaaaag ccaaccatca
     2161 tgctattcca tccaaaacga ttttcggtaa atagcaccca caccgttgcg ggaatttggc
     2221 ctatcaattg cgctgaaaaa taaataatca acaaaatggg catcgtttta aataaagtga
     2281 tgtacaccga atttgattgc gtctcaaccc ctacttcggt atctgtatta tcacgtgtat
     2341 ttttggtttc acggaaccaa aacataacca caaggaaagt gacaatattt agcaacgcag
     2401 cgataaaaaa gggactatgc ggtgaaatct ctcctgcaaa accaccaata ataggccccg
     2461 ctattaaacc aagcccaaaa cttgccccta accaaccgaa ccacttcacg cgttgagaag
     2521 ctgaggtggt atcggcaatg accgatgccg cgacagcccc agtagctcct gtgatccctg
     2581 aaagcaaacg gcctaaatac agcatccaaa gcgcacttga aaaagccagc aataagtaat
     2641 ccagcgatgc gcctattaat gacaacaaca gcactgggcg ccgaccaaat cggtcagaca
     2701 tttttccaag ccaaggagca aagataacct gcattaacgc ataaagtgca agcaatacgc
     2761 caaagtggtt agcgatatct tccgaagcaa taaattcacg taataacgtt ggcaagactg
     2821 gcatgataag gccaatcccc atggcatcga gcaacgtaat taccaatgcg atctttgtcg
     2881 aactattcat ttcacttttc tctatcactg atagggagtg ggaaaataac tctatcaatg
     2941 atagggtgtc aaatcgatgc acaacgtgcg ttacatatca aagggaaaac tgtccatacc
     3001 catggtaagt aggtcattat tagtcaaaat aaaatcattt gtcgatttca attttgtccc
     3061 attcccgtcc acggctgtcc ctataccgcg ccgccattga atctgattta tgcccgagaa
     3121 gacgttgagc aaacttatcg ccaatctggt tccggtatag cctcgctgac aggctacgca
     3181 gttcatggaa tgttggcggg tttccatcaa atgagagtcc agatgcattt ctcgcctttg
     3241 taaaatactt tgatactgtt ttcggggaaa gcggatcgtg atgctttgat gcgattatag
     3301 tttcactgct gctggcctcc ctgcattgct gtagtgtatc agccaatgag atattgagcg
     3361 cgtcaatcgt tagcgttagt ggaatggcga gtttagcccc tgttttactc tgttcaatgt
     3421 gaagatggtt gtcgtttatg tctgaccatt tcattctgca caaatcgcct actctctgcc
     3481 ctgtaacgac agccaaatcc attgccagtc tcagccagat tgggagcggt tcggctgcat
     3541 ggtatatcgc gacatattca ttagctgtca gtcttgaacg ccttacttct gattttgctg
     3601 tgcgggttgc tgttaccgga ttcgtagcca catgcccctc ggctattgcc tcacgaaaaa
     3661 cgtcaacaag ggttgacctg atcaattttg cggaagccga tttgccttct gctacgtagg
     3721 tgtttagcat tgctgccacc tctttcgttg atatgtcagc gagcggtttg tccggcaatt
     3781 ttcttcggat tgctctgatt ttgctggcgt agtcgagtag agttttcggc ctgatacccc
     3841 tctcgctgag gattgtttca tatcggtcaa gccacgcatg aagagtgatt gcgtcagcgc
     3901 ctttaattct gtctatcagt gactcacgcc tgttcccgga tagcaactca atgttggcct
     3961 gtatagcttc agtgattgcg attcgcctgt ctcggcctaa tccaaactct ttacccgtcc
     4021 ttgggtccct gtagcagtaa tatccattgt ttcttatata aaggttaggg ggtaaatccc
     4081 ggcgctcatg acttcgcctt cttcccattt ctgatcctct tcaaaaggct acacgcgtat
     4141 acaacctcct tagtacatgc aaccattatc accgccagag gtaaaatagt caacacgcac
     4201 ggtgttagat atttatccct tgcggtgata gatttaacgt atgagcacaa aaaagaaacc
     4261 attaacacaa gagcagcttg aggacgcacg tcgccttaaa gcaatttatg aaaaaaagaa
     4321 aaatgaactt ggcttatccc aggaatctgt cgcagacaag atggggatgg ggcagtcagg
     4381 cgttggtgct ttatttaatg gcatcaatgc attaaatgct tataacgccg cattgcttac
     4441 aaaaattctc aaagttagcg ttgaagaatt tagcccttca atcgccagag aaatctacga
     4501 gatgtatgaa gcggttagta tgcagccgtc acttagaagt gagtatgagt accctgtttt
     4561 ttctcatgtt caggcaggga tgttctcacc taagcttaga acctttacca aaggtgatgc
     4621 ggagagatgg gtaagcacaa ccaaaaaagc cagtgattct gcattctggc ttgaggttga
     4681 aggtaattcc atgaccgcac caacaggctc caagccaagc tttcctgacg gaatgttaat
     4741 tctcgttgac cctgagcagg ctgttgagcc aggtgatttc tgcatagcca gacttggggg
     4801 tgatgagttt accttcaaga aactgatcag ggatagcggt caggtgtttt tacaaccact
     4861 aaacccacag tacccaatga tcccatgcaa tgagagttgt tccgttgtgg ggaaagttat
     4921 cgctagtcag tggcctgaag agacgtttgg ctgacctagg gaagttccta tactttctag
     4981 agaataggaa cttcggacgt ctaagaaacc attattatca tgagggccca ccggttaatt
     5041 aacggcacca ccgaaattca gtaagcagaa agtcaaaagc ctccgaccgg aggcttttga
     5101 ctattactca acaggtaagg cgcgaggttt tttcaattgt tcgaaacgca aaaaggccat
     5161 ccgtcaggat ggccttctgc ttaatttgat gcctgtcagt ttaggttagg cgccatgcat
     5221 ctcgaggcat gcctgcagcg gccgctacta gtaatgtgag caaaaggcca gcaaaaggcc
     5281 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5341 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5401 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5461 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5521 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5581 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5641 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5701 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     5761 tttggtatct gcgctctgct aaagccagtt accttcggaa aaagagttgg tagctcttga
     5821 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagta gcagattacg
     5881 cgcagaaaaa aaggatctca agaagatctt ttgatctttt ctacgggtta ttatattccc
     5941 cagaacatca ggttaatggc gtttttgatg tcattttcgc ggtggctgag atcagccact
     6001 tcttccccga taacggagac cggcacactg gccatatcgg tggtcatcat gcgccagctt
     6061 tcatccccga tatgcaccac cgggtaaagt tcacgggaga ctttatctga cagcagacgt
     6121 gcactggcca gggggatcac catccgtcgc ccgggcgtgt caataatatc actctgtaca
     6181 tccacaaaca gacgataacg gctctctctt ttataggtgt aaaccttaaa ctgcatagca
     6241 tacctctttt tgacatactt cgggtataca tatcagtata tattcttata ccgcaaaaat
     6301 cagcgcgcaa atacgcatac tgttatctgg cttttagtaa gccctctaga ggatccccgg
     6361 gtaccgagct cgaattccga tccccaattc ctggcagttt atggcgggcg tcctgcccgc
     6421 caccctccgg gccgttgctt cgcaacgttc aaatccgctc ccggcggatt tgtcctactc
     6481 aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc
     6541 tttcgtttta tttgatgcct ggggaattaa ttcctggcag tttatggcgg gcgtcctgcc
     6601 cgccaccctc cgggccgttg cttcgcaacg ttcaaatccg ctcccggcgg atttgtccta
     6661 ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga
     6721 gcctttcgtt ttatttgatg cctggggaat t
//
