LOCUS       sequence-327715-        6694 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 6694)
  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 6694)
  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-CT (CamR, P21)
FEATURES             Location/Qualifiers
     source          1..6694
                     /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             2299..3441
                     /codon_start=1
                     /gene="int"
                     /product="integrase from phage phi-21"
                     /label=phage phi-21 integrase
                     /translation="MAARPRSHKISIPNLYCKLDKRTGKVYWQYKHPLSGRFHSLGTDE
                     NEAKQVATEANTIIAEQRTRQILSVNERLERMKGRRSDITVTEWLDKYISIQEDRLQHN
                     ELRPNSYRQKGKPIRLFREHCGMQHLKDITALDIAEIIDAVKAEGHNRMAQVVRMVLID
                     VFKEAQHAGHVPPGFNPAQATKQPRNRVNRQRLSLLEWQAIFESVSRRQPYLKCGMLLA
                     LVTGQRLGDICNLKFSDIWDDMLHITQEKTGSKLAIPLNLKCDALNITLREVISQCRDA
                     VVSKYLVHYRHTTSQANRGDQVSANTLTTAFKKAKEKCGIKWEQGTAPTFHEQRSLSER
                     LYREQGLDTQKLLGHKSRKMTDRYNDDRGKDWIIVDIKTA"
     promoter        3603..3705
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3706..4365
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3772..3791)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      4386..4480
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4537..4925
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    4936..4983
                     /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    5063..5562
                     /label=phage phi-21 attP
                     /bound_moiety="phage phi-21 integrase"
                     /note="attachment site of phage phi-21"
     terminator      complement(5583..5829)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(5920..5942)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6093..6111
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      6152..6238
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6335..6421
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6518..6604
                     /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 cgcgtagaac cgcaactccc aataaacgca aacccaaaac
     2281 tccaacggat aatcgctgat ggctgctaga ccccgatctc acaaaatctc tatacccaat
     2341 ttatattgca aattagataa gcgaaccgga aaggtatatt ggcaatacaa acatccacta
     2401 tccggtcgtt ttcatagctt aggaactgat gagaatgaag caaaacaagt tgctactgaa
     2461 gcaaatacca ttattgctga acaacgtacc agacaaatat taagcgtcaa tgagcgtctg
     2521 gaaagaatga aaggcaggcg ctcagacatt acggtgacag aatggcttga taaatatatt
     2581 tctatccagg aggacaggct gcaacataat gaactaagac ccaactccta tcggcaaaaa
     2641 ggcaaaccca ttcgtctttt ccgtgagcat tgtggaatgc aacacctcaa ggatattacc
     2701 gcacttgata ttgccgaaat aattgatgct gtaaaggctg aaggtcataa caggatggcg
     2761 caagtcgtga gaatggtgtt gatcgacgtc ttcaaagaag cacaacacgc aggacatgtt
     2821 ccgccaggat ttaacccagc gcaggcaaca aaacaaccgc gaaatcgagt aaaccgccaa
     2881 agattgtcac tgctcgaatg gcaggcaata tttgaaagcg taagcagacg gcagccctat
     2941 ttaaaatgcg gcatgctact tgctcttgtt actggacaac gtttaggcga tatctgcaat
     3001 ttgaaattct ctgatatatg ggacgacatg ttgcacatta ctcaggaaaa aaccggttca
     3061 aaacttgcta ttccgcttaa cctgaaatgc gatgctctga atattaccct tcgtgaagtt
     3121 atatctcagt gcagggatgc tgttgttagt aaatatctgg tccattaccg tcacactacc
     3181 tctcaagcaa acagaggaga ccaggtttct gcaaatactc tgacaacggc ttttaaaaag
     3241 gccaaggaaa aatgtggcat aaaatgggag caaggaactg cgcccacatt tcatgaacag
     3301 cgatctctgt cagaacgatt atatcgggaa cagggtctgg atacgcaaaa gttgttaggc
     3361 cataaatcca gaaaaatgac cgaccgatac aatgatgatc gtggtaaaga ctggattatc
     3421 gtagatatca aaacagcata gaaaatagcc agttttgggg aagggttttg gggaaagttt
     3481 tggggaagat tttacatcat cataaaacaa cgggcgtgtt atacgcccgt tatccatggg
     3541 tatggacagt tttccctttg atatgtaacg cacgttgtgg atatctggcg aaaatgagac
     3601 gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc
     3661 gtattttttg agttatcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc
     3721 actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt
     3781 cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta
     3841 aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc
     3901 ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg
     3961 gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc
     4021 tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg
     4081 tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc
     4141 tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac
     4201 ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg
     4261 ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt
     4321 aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaatttga tatcgagctc
     4381 gcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag
     4441 aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaagcgtcg acctagtgcg
     4501 gccgcaagat ccggccacga tgcgtccggc gtagaggatc tgaagatcag cagttcaacc
     4561 tgttgatagt acgtactaag ctctcatgtt tcacgtacta agctctcatg tttaacgtac
     4621 taagctctca tgtttaacga actaaaccct catggctaac gtactaagct ctcatggcta
     4681 acgtactaag ctctcatgtt tcacgtacta agctctcatg tttgaacaat aaaattaata
     4741 taaatcagca acttaaatag cctctaaggt tttaagtttt ataagaaaaa aaagaatata
     4801 taaggctttt aaagctttta aggtttaacg gttgtggaca acaagccagg gatgtaacgc
     4861 actgagaagc ccttagagcc tctcaaagca attttgagtg acacaggaac acttaacggc
     4921 tgacatggga attaggaagt tcctattccg aagttcctat tctctagaaa gtataggaac
     4981 ttccatatgc catggaatga ccgaccgata caatgatgat cgtggtaaag actggattat
     5041 cgtagatatc aaaacagcat agaaaatagc cagttttggg gaagggtttt ggggaaagtt
     5101 ttggggaaga ttttacatca tcataaaaca acgggcgtgt tatacgcccg ttataatatt
     5161 taacacatgt ggtaattaca tatttttgat gatcgcgtca ccaaactcgc tgcatttcag
     5221 cagtttagcg ccttccatca gacgttcgaa gtcataggtt acggtcttgg cgttgattgc
     5281 gccttccata cctttaacaa tcaggtctgc ggcttcagtc cagcccatat ggcgcagcag
     5341 tatataaaaa ttatgtattc aactcgctga tatttaatca taaatattat tttcttgtct
     5401 ttgtattagc caaaatcaac tttattcaac cgtttgattt cattgacgtt cgtttttatt
     5461 ttggggaaag attttctcta ccatttatat caaaattaca agtctacatt ttattaacat
     5521 ctcaatacat gtgtggcctt gcaagccatt gcgaggcctt atgctagctt cttcgtctgt
     5581 ttctactggt attggcacaa acctgattcc aatttgagca aggctatgtg ccatctcgat
     5641 actcgttctt aactcaacag aagatgcttt gtgcatacag cccctcgttt attatttatc
     5701 tcctcagcca gccgctgtgc tttcagtgga tttcggataa cagaaaggcc gggaaatacc
     5761 cagcctcgct ttgtaacgga gtagagacga aagtgattgc gcctacccgg atattatcgt
     5821 gaggatgcgt catcgccatt aattcactga tcagtgataa gctgtcaaac atgagaattg
     5881 atccggctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     5941 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     6001 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     6061 agtgtatgct gcacatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     6121 ttcaagaatt aattcccaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     6181 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     6241 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc
     6301 gccataaact gccaggaatt aattccccag gcatcaaata aaacgaaagg ctcagtcgaa
     6361 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     6421 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     6481 cccgccataa actgccagga attaattccc caggcatcaa ataaaacgaa aggctcagtc
     6541 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     6601 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
     6661 acgcccgcca taaactgcca ggaattgggg atcg
//
