LOCUS       sequence-292133-        6224 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 6224)
  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 6224)
  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-CH (CamR, HK022)
FEATURES             Location/Qualifiers
     source          1..6224
                     /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"
     promoter        3442..3544
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3545..4204
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3611..3630)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      4225..4319
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4376..4764
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    4775..4822
                     /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    4852..5078
                     /gene="attP"
                     /label=HK022 attP
                     /bound_moiety="HK022 integrase"
                     /note="attachment site of phage HK022"
     terminator      complement(5113..5359)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(5450..5472)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5623..5641
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      5682..5768
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5865..5951
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6048..6134
                     /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 acgttgtgga
     3421 tatctggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga
     3481 aataagatca ctaccgggcg tattttttga gttatcgaga ttttcaggag ctaaggaagc
     3541 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa
     3601 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
     3661 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt
     3721 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga
     3781 cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac
     3841 tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat
     3901 atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat
     3961 tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa
     4021 cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca
     4081 aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt
     4141 ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc
     4201 gtaatttgat atcgagctcg cttggactcc tgttgataga tccagtaatg acctcagaac
     4261 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc
     4321 caagcgtcga cctagtgcgg ccgcaagatc cggccacgat gcgtccggcg tagaggatct
     4381 gaagatcagc agttcaacct gttgatagta cgtactaagc tctcatgttt cacgtactaa
     4441 gctctcatgt ttaacgtact aagctctcat gtttaacgaa ctaaaccctc atggctaacg
     4501 tactaagctc tcatggctaa cgtactaagc tctcatgttt cacgtactaa gctctcatgt
     4561 ttgaacaata aaattaatat aaatcagcaa cttaaatagc ctctaaggtt ttaagtttta
     4621 taagaaaaaa aagaatatat aaggctttta aagcttttaa ggtttaacgg ttgtggacaa
     4681 caagccaggg atgtaacgca ctgagaagcc cttagagcct ctcaaagcaa ttttgagtga
     4741 cacaggaaca cttaacggct gacatgggaa ttaggaagtt cctattccga agttcctatt
     4801 ctctagaaag tataggaact tccatatgcc atgggacaaa attgaaatcg acaaatgatt
     4861 ttattttgac taataatgac ctacttacat taatttactg ataattaaag agattttaaa
     4921 tatacaactt attcacctaa aggatgacaa aataacatta atcacttaaa aatcatcgca
     4981 ttacactaat ctgtggttaa atgatagact acataatgcg acaaaacgca acatatccag
     5041 tcactatgaa tcaactactt agatagtatt agtgacctga gacagagcat tagctagctt
     5101 cttcgtctgt ttctactggt attggcacaa acctgattcc aatttgagca aggctatgtg
     5161 ccatctcgat actcgttctt aactcaacag aagatgcttt gtgcatacag cccctcgttt
     5221 attatttatc tcctcagcca gccgctgtgc tttcagtgga tttcggataa cagaaaggcc
     5281 gggaaatacc cagcctcgct ttgtaacgga gtagagacga aagtgattgc gcctacccgg
     5341 atattatcgt gaggatgcgt catcgccatt aattcactga tcagtgataa gctgtcaaac
     5401 atgagaattg atccggctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     5461 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     5521 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     5581 gcgatagcgg agtgtatgct gcacatgaca ttaacctata aaaataggcg tatcacgagg
     5641 ccctttcgtc ttcaagaatt aattcccaat tccccaggca tcaaataaaa cgaaaggctc
     5701 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     5761 ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg
     5821 caggacgccc gccataaact gccaggaatt aattccccag gcatcaaata aaacgaaagg
     5881 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
     5941 gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc
     6001 gggcaggacg cccgccataa actgccagga attaattccc caggcatcaa ataaaacgaa
     6061 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     6121 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     6181 ggcgggcagg acgcccgcca taaactgcca ggaattgggg atcg
//
