LOCUS       sequence-352785-        7023 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 7023)
  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 7023)
  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-TT (TetR, P21)
FEATURES             Location/Qualifiers
     source          1..7023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(47..133)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(174..192)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     343..365
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     terminator      456..702
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     protein_bind    723..1222
                     /label=phage phi-21 attP
                     /bound_moiety="phage phi-21 integrase"
                     /note="attachment site of phage phi-21"
     protein_bind    complement(1302..1349)
                     /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      1360..1748
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(1805..1899)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1958..3163)
                     /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        3171..3225
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    3172..3190
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             complement(3371..4513)
                     /codon_start=1
                     /gene="int"
                     /product="integrase from phage phi-21"
                     /label=phage phi-21 integrase
                     /translation="MAARPRSHKISIPNLYCKLDKRTGKVYWQYKHPLSGRFHSLGTDE
                     NEAKQVATEANTIIAEQRTRQILSVNERLERMKGRRSDITVTEWLDKYISIQEDRLQHN
                     ELRPNSYRQKGKPIRLFREHCGMQHLKDITALDIAEIIDAVKAEGHNRMAQVVRMVLID
                     VFKEAQHAGHVPPGFNPAQATKQPRNRVNRQRLSLLEWQAIFESVSRRQPYLKCGMLLA
                     LVTGQRLGDICNLKFSDIWDDMLHITQEKTGSKLAIPLNLKCDALNITLREVISQCRDA
                     VVSKYLVHYRHTTSQANRGDQVSANTLTTAFKKAKEKCGIKWEQGTAPTFHEQRSLSER
                     LYREQGLDTQKLLGHKSRKMTDRYNDDRGKDWIIVDIKTA"
     RBS             4578..4586
                     /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             4675..5388
                     /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(5395..5428)
                     /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      5505..5536
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     terminator      complement(5585..5612)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     misc_feature    5667..5687
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     rep_origin      complement(5747..6335)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5827..5846)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6365..6670)
                     /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(6467..6486)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      complement(6902..6988)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 accctccggg ccgttgcttc gcaacgttca aatccgctcc cggcggattt gtcctactca
       61 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct
      121 ttcgttttat ttgatgcctg gggaattggg aattaattct tgaagacgaa agggcctcgt
      181 gatacgccta tttttatagg ttaatgtcat gtgcagcata cactccgcta tcgctacgtg
      241 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
      301 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
      361 agaggttttc accgtcatca ccgaaacgcg cgaggcagcc ggatcaattc tcatgtttga
      421 cagcttatca ctgatcagtg aattaatggc gatgacgcat cctcacgata atatccgggt
      481 aggcgcaatc actttcgtct ctactccgtt acaaagcgag gctgggtatt tcccggcctt
      541 tctgttatcc gaaatccact gaaagcacag cggctggctg aggagataaa taataaacga
      601 ggggctgtat gcacaaagca tcttctgttg agttaagaac gagtatcgag atggcacata
      661 gccttgctca aattggaatc aggtttgtgc caataccagt agaaacagac gaagaagcta
      721 gcataaggcc tcgcaatggc ttgcaaggcc acacatgtat tgagatgtta ataaaatgta
      781 gacttgtaat tttgatataa atggtagaga aaatctttcc ccaaaataaa aacgaacgtc
      841 aatgaaatca aacggttgaa taaagttgat tttggctaat acaaagacaa gaaaataata
      901 tttatgatta aatatcagcg agttgaatac ataattttta tatactgctg cgccatatgg
      961 gctggactga agccgcagac ctgattgtta aaggtatgga aggcgcaatc aacgccaaga
     1021 ccgtaaccta tgacttcgaa cgtctgatgg aaggcgctaa actgctgaaa tgcagcgagt
     1081 ttggtgacgc gatcatcaaa aatatgtaat taccacatgt gttaaatatt ataacgggcg
     1141 tataacacgc ccgttgtttt atgatgatgt aaaatcttcc ccaaaacttt ccccaaaacc
     1201 cttccccaaa actggctatt ttctatgctg ttttgatatc tacgataatc cagtctttac
     1261 cacgatcatc attgtatcgg tcggtcattc catggcatat ggaagttcct atactttcta
     1321 gagaatagga acttcggaat aggaacttcc taattcccat gtcagccgtt aagtgttcct
     1381 gtgtcactca aaattgcttt gagaggctct aagggcttct cagtgcgtta catccctggc
     1441 ttgttgtcca caaccgttaa accttaaaag ctttaaaagc cttatatatt cttttttttc
     1501 ttataaaact taaaacctta gaggctattt aagttgctga tttatattaa ttttattgtt
     1561 caaacatgag agcttagtac gtgaaacatg agagcttagt acgttagcca tgagagctta
     1621 gtacgttagc catgagggtt tagttcgtta aacatgagag cttagtacgt taaacatgag
     1681 agcttagtac gtgaaacatg agagcttagt acgtactatc aacaggttga actgctgatc
     1741 ttcagatcct ctacgccgga cgcatcgtgg ccggatcttg cggccgcact aggtcgacgc
     1801 ttggattctc accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat
     1861 ggagttctga ggtcattact ggatctatca acaggagtcc aagcgagctc aaaaattaaa
     1921 aatgaagttt tggaggcctc atttggtgac gaaataacta agcacttgtc tcctgtttac
     1981 tcccctgagc ttgaggggtc aacatgaagg tcattgatag caggataata atacagtaaa
     2041 acgctaaacc aataatccaa atccagccat cccaaattgg tagtgaatga ttataaataa
     2101 cagcaaacag taatgggcca ataacaccgg ttgcattggt aaggctcacc aataatccct
     2161 gtaaagcacc ttgctgatga ctctttgttt ggatagacat cactccctgt aatgcaggta
     2221 aagcgatccc accaccagcc aataaaatta aaacagggaa aactaaccaa ccttcagata
     2281 taaacgctaa aaaggcaaat gcactactat ctgcaataaa tccgagcagt actgccgttt
     2341 tttcgcccca tttagtggct attcttcctg ccacaaaggc ttggaatact gagtgtaaaa
     2401 gaccaagacc cgctaatgaa aagccaacca tcatgctatt ccatccaaaa cgattttcgg
     2461 taaatagcac ccacaccgtt gcgggaattt ggcctatcaa ttgcgctgaa aaataaataa
     2521 tcaacaaaat gggcatcgtt ttaaataaag tgatgtacac cgaatttgat tgcgtctcaa
     2581 cccctacttc ggtatctgta ttatcacgtg tatttttggt ttcacggaac caaaacataa
     2641 ccacaaggaa agtgacaata tttagcaacg cagcgataaa aaagggacta tgcggtgaaa
     2701 tctctcctgc aaaaccacca ataataggcc ccgctattaa accaagccca aaacttgccc
     2761 ctaaccaacc gaaccacttc acgcgttgag aagctgaggt ggtatcggca atgaccgatg
     2821 ccgcgacagc cccagtagct cctgtgatcc ctgaaagcaa acggcctaaa tacagcatcc
     2881 aaagcgcact tgaaaaagcc agcaataagt aatccagcga tgcgcctatt aatgacaaca
     2941 acagcactgg gcgccgacca aatcggtcag acatttttcc aagccaagga gcaaagataa
     3001 cctgcattaa cgcataaagt gcaagcaata cgccaaagtg gttagcgata tcttccgaag
     3061 caataaattc acgtaataac gttggcaaga ctggcatgat aaggccaatc cccatggcat
     3121 cgagcaacgt aattaccaat gcgatctttg tcgaactatt catttcactt ttctctatca
     3181 ctgataggga gtgggaaaat aactctatca atgatagggt gtcaaatcga tgcacaacgt
     3241 gcgttacata tcaaagggaa aactgtccat acccatggat aacgggcgta taacacgccc
     3301 gttgttttat gatgatgtaa aatcttcccc aaaactttcc ccaaaaccct tccccaaaac
     3361 tggctatttt ctatgctgtt ttgatatcta cgataatcca gtctttacca cgatcatcat
     3421 tgtatcggtc ggtcattttt ctggatttat ggcctaacaa cttttgcgta tccagaccct
     3481 gttcccgata taatcgttct gacagagatc gctgttcatg aaatgtgggc gcagttcctt
     3541 gctcccattt tatgccacat ttttccttgg cctttttaaa agccgttgtc agagtatttg
     3601 cagaaacctg gtctcctctg tttgcttgag aggtagtgtg acggtaatgg accagatatt
     3661 tactaacaac agcatccctg cactgagata taacttcacg aagggtaata ttcagagcat
     3721 cgcatttcag gttaagcgga atagcaagtt ttgaaccggt tttttcctga gtaatgtgca
     3781 acatgtcgtc ccatatatca gagaatttca aattgcagat atcgcctaaa cgttgtccag
     3841 taacaagagc aagtagcatg ccgcatttta aatagggctg ccgtctgctt acgctttcaa
     3901 atattgcctg ccattcgagc agtgacaatc tttggcggtt tactcgattt cgcggttgtt
     3961 ttgttgcctg cgctgggtta aatcctggcg gaacatgtcc tgcgtgttgt gcttctttga
     4021 agacgtcgat caacaccatt ctcacgactt gcgccatcct gttatgacct tcagccttta
     4081 cagcatcaat tatttcggca atatcaagtg cggtaatatc cttgaggtgt tgcattccac
     4141 aatgctcacg gaaaagacga atgggtttgc ctttttgccg ataggagttg ggtcttagtt
     4201 cattatgttg cagcctgtcc tcctggatag aaatatattt atcaagccat tctgtcaccg
     4261 taatgtctga gcgcctgcct ttcattcttt ccagacgctc attgacgctt aatatttgtc
     4321 tggtacgttg ttcagcaata atggtatttg cttcagtagc aacttgtttt gcttcattct
     4381 catcagttcc taagctatga aaacgaccgg atagtggatg tttgtattgc caatatacct
     4441 ttccggttcg cttatctaat ttgcaatata aattgggtat agagattttg tgagatcggg
     4501 gtctagcagc catcagcgat tatccgttgg agttttgggt ttgcgtttat tgggagttgc
     4561 ggttctacgc gtatacaacc tccttagtac atgcaaccat tatcaccgcc agaggtaaaa
     4621 tagtcaacac gcacggtgtt agatatttat cccttgcggt gatagattta acgtatgagc
     4681 acaaaaaaga aaccattaac acaagagcag cttgaggacg cacgtcgcct taaagcaatt
     4741 tatgaaaaaa agaaaaatga acttggctta tcccaggaat ctgtcgcaga caagatgggg
     4801 atggggcagt caggcgttgg tgctttattt aatggcatca atgcattaaa tgcttataac
     4861 gccgcattgc ttacaaaaat tctcaaagtt agcgttgaag aatttagccc ttcaatcgcc
     4921 agagaaatct acgagatgta tgaagcggtt agtatgcagc cgtcacttag aagtgagtat
     4981 gagtaccctg ttttttctca tgttcaggca gggatgttct cacctaagct tagaaccttt
     5041 accaaaggtg atgcggagag atgggtaagc acaaccaaaa aagccagtga ttctgcattc
     5101 tggcttgagg ttgaaggtaa ttccatgacc gcaccaacag gctccaagcc aagctttcct
     5161 gacggaatgt taattctcgt tgaccctgag caggctgttg agccaggtga tttctgcata
     5221 gccagacttg ggggtgatga gtttaccttc aagaaactga tcagggatag cggtcaggtg
     5281 tttttacaac cactaaaccc acagtaccca atgatcccat gcaatgagag ttgttccgtt
     5341 gtggggaaag ttatcgctag tcagtggcct gaagagacgt ttggctgacc tagggaagtt
     5401 cctatacttt ctagagaata ggaacttcgg acgtctaaga aaccattatt atcatgaggg
     5461 cccaccggtt aattaacggc accaccgaaa ttcagtaagc agaaagtcaa aagcctccga
     5521 ccggaggctt ttgactatta ctcaacaggt aaggcgcgag gttttttcaa ttgttcgaaa
     5581 cgcaaaaagg ccatccgtca ggatggcctt ctgcttaatt tgatgcctgt cagtttaggt
     5641 taggcgccat gcatctcgag gcatgcctgc agcggccgct actagtaatg tgagcaaaag
     5701 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5761 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5821 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5881 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5941 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6001 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6061 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6121 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6181 ctagaaggac agtatttggt atctgcgctc tgctaaagcc agttaccttc ggaaaaagag
     6241 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6301 agtagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcttttgatc ttttctacgg
     6361 gttattatat tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc
     6421 tgagatcagc cacttcttcc ccgataacgg agaccggcac actggccata tcggtggtca
     6481 tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat
     6541 ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa
     6601 tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct
     6661 taaactgcat agcatacctc tttttgacat acttcgggta tacatatcag tatatattct
     6721 tataccgcaa aaatcagcgc gcaaatacgc atactgttat ctggctttta gtaagccctc
     6781 tagaggatcc ccgggtaccg agctcgaatt ccgatcccca attcctggca gtttatggcg
     6841 ggcgtcctgc ccgccaccct ccgggccgtt gcttcgcaac gttcaaatcc gctcccggcg
     6901 gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt
     6961 ctttcgactg agcctttcgt tttatttgat gcctggggaa ttaattcctg gcagtttatg
     7021 gcg
//
