LOCUS       sequence-219871-        6345 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 6345)
  AUTHORS   Donnenberg MS, Kaper JB
  TITLE     Construction of an eae deletion mutant of enteropathogenic 
            Escherichia coli by using a positive-selection suicide vector.
  JOURNAL   Infect Immun. 1991 Dec . 59(12):4310-7.
  PUBMED    1937792
REFERENCE   2  (bases 1 to 6345)
  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"; journalName: "Infect 
            Immun. 1991 Dec . 59(12):4310-7."
COMMENT     Sequence Label: pCVD442
FEATURES             Location/Qualifiers
     source          1..6345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     143..162
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     oriT            843..952
                     /note="incP origin of transfer"
     CDS             985..1356
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     rep_origin      1974..2362
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    2397..2413
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        2539..2984
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(2985..3071,3072..4406)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        5145..5249
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5250..5318,5319..6110)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5468..5487)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(6167..6186)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(6327..6345,1..4)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gaggttttca ccgtcatcac cgaaacgcgc gaggcagcaa ggagatggcg cccaacagtc
       61 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
      121 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
      181 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatccttttt gtccggtgtt
      241 gggttgaagg tgaagccggt cggggccgca gcgggggccg gcttttcagc cttgcccccc
      301 tgcttcggcc gccgtggctc cggcgtcttg ggtgccggcg cgggttccgc agccttggcc
      361 tgcggtgcgg gcacatcggc gggcttggcc ttgatgtgcc gcctggcgtg cgagcggaac
      421 gtctcgtagg agaacttgac cttccccgtt tcccgcatgt gctcccaaat ggtgacgagc
      481 gcatagccgg acgctaacgc cgcctcgaca tccgccctca ccgccaggaa cgcaaccgca
      541 gcctcatcac gccggcgctt cttggccgcg cgggattcaa cccactcggc cagctcgtcg
      601 gtgtagctct ttggcatcgt ctctcgcctg tcccctcagt tcagtaattt cctgcatttg
      661 cctgtttcca gtcggtagat attccacaaa acagcaggga agcagcgctt ttccgctgca
      721 taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga
      781 tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag
      841 ccgggcagga taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct
      901 tattcgcacc tggcggtgct caacgggaat cctgctctgc gaggctggcc ggctaccgcc
      961 ggcgtaacag atgagggcaa gcggatggct gatgaaacca agccaaccag gaagggcagc
     1021 ccacctatca aggtgtactg ccttccagac gaacgaagag cgattgagga aaaggcggcg
     1081 gcggccggca tgagcctgtc ggcctacctg ctggccgtcg gccagggcta caaaatcacg
     1141 ggcgtcgtgg actatgagca cgtccgcgag ctggcccgca tcaatggcga cctgggccgc
     1201 ctgggcggcc tgctgaaact ctggctcacc gacgacccgc gcacggcgcg gttcggtgat
     1261 gccacgatcc tcgccctgct ggcgaagatc gaagagaagc aggacgagct tggcaaggtc
     1321 atgatgggcg tggtccgccc gagggcagag ccatgacttt tttagccgct aaaacggccg
     1381 gggggtgcgc gtgattgcca agcacgtccc catgcgctcc atcaagaaga gcgacttcgc
     1441 ggagctggtg aagtacatca ccgacgagca aggcaagacc gagcgcctgg gtcacgtgcg
     1501 cgtcacgaac tgcgaggcaa acaccctgcc cgctgtcatg gccgaggtga tggcgaccca
     1561 gcacggcaac acccgttccg aggccgacaa gacctatcac ctgctggtta gcttccgcgc
     1621 gggagagaag cccgacgcgg agacgttgcg cgcgattgag gaccgcatct gcgctgggct
     1681 tggcttcgcc gagcatcagc gcgtcagtgc cgtgcatcac gacaccgaca acctgcacat
     1741 ccatatcgcc atcaacaaga ttcacccgac ccgaaacacc atccatgagc cgtatcgggc
     1801 ctaccgcgcc ctcgctgacc tctgcgcgac gctcgaacgg gactacgggc ttgagcgtga
     1861 caatcacgaa acgcggcagc gcgtttccga gaaccgcgcg aacgacatgg agcggcacgc
     1921 gggcgtggaa agcctggtcg gctggatccg gccacgatgc gtccggcgta gaggatctga
     1981 agatcagcag ttcaacctgt tgatagtacg tactaagctc tcatgtttca cgtactaagc
     2041 tctcatgttt aacgtactaa gctctcatgt ttaacgaact aaaccctcat ggctaacgta
     2101 ctaagctctc atggctaacg tactaagctc tcatgtttca cgtactaagc tctcatgttt
     2161 gaacaataaa attaatataa atcagcaact taaatagcct ctaaggtttt aagttttata
     2221 agaaaaaaaa gaatatataa ggcttttaaa gcttttaagg tttaacggtt gtggacaaca
     2281 agccagggat gtaacgcact gagaagccct tagagcctct caaagcaatt ttgagtgaca
     2341 caggaacact taacggctga catgggaatt ccacatgtgg aattccacat gtggaattgt
     2401 gagcggataa caatttgtgg aattcccggg agagctcgat atcgcatgcg gtacctctag
     2461 aagaagcttg ggatccgtcg acctgcagat ccgtcgacct gcaggtcgac tctagaggat
     2521 cgatcctttt taacccatca catatacctg ccgttcacta ttatttagtg aaatgagata
     2581 ttatgatatt ttctgaattg tgattaaaaa ggcaacttta tgcccatgca acagaaacta
     2641 taaaaaatac agagaatgaa aagaaacaga tagatttttt agttctttag gcccgtagtc
     2701 tgcaaatcct tttatgattt tctatcaaac aaaagaggaa aatagaccag ttgcaatcca
     2761 aacgagagtc taatagaatg aggtcgaaaa gtaaatcgcg cgggtttgtt actgataaag
     2821 caggcaagac ctaaaatgtg taaagggcaa agtgtatact ttggcgtcac cccttacata
     2881 ttttaggtct ttttttattg tgcgtaacta acttgccatc ttcaaacagg agggctggaa
     2941 gaagcagacc gctaacacag tacataaaaa aggagacatg aacgatgaac atcaaaaagt
     3001 ttgcaaaaca agcaacagta ttaaccttta ctaccgcact gctggcagga ggcgcaactc
     3061 aagcgtttgc gaaagaaacg aaccaaaagc catataagga aacatacggc atttcccata
     3121 ttacacgcca tgatatgctg caaatccctg aacagcaaaa aaatgaaaaa tatcaagttc
     3181 ctgaattcga ttcgtccaca attaaaaata tctcttctgc aaaaggcctg gacgtttggg
     3241 acagctggcc attacaaaac gctgacggca ctgtcgcaaa ctatcacggc taccacatcg
     3301 tctttgcatt agccggagat cctaaaaatg cggatgacac atcgatttac atgttctatc
     3361 aaaaagtcgg cgaaacttct attgacagct ggaaaaacgc tggccgcgtc tttaaagaca
     3421 gcgacaaatt cgatgcaaat gattctatcc taaaagacca aacacaagaa tggtcaggtt
     3481 cagccacatt tacatctgac ggaaaaatcc gtttattcta cactgatttc tccggtaaac
     3541 attacggcaa acaaacactg acaactgcac aagttaacgt atcagcatca gacagctctt
     3601 tgaacatcaa cggtgtagag gattataaat caatctttga cggtgacgga aaaacgtatc
     3661 aaaatgtaca gcagttcatc gatgaaggca actacagctc aggcgacaac catacgctga
     3721 gagatcctca ctacgtagaa gataaaggcc acaaatactt agtatttgaa gcaaacactg
     3781 gaactgaaga tggctaccaa ggcgaagaat ctttatttaa caaagcatac tatggcaaaa
     3841 gcacatcatt cttccgtcaa gaaagtcaaa aacttctgca aagcgataaa aaacgcacgg
     3901 ctgagttagc aaacggcgct ctcggtatga ttgagctaaa cgatgattac acactgaaaa
     3961 aagtgatgaa accgctgatt gcatctaaca cagtaacaga tgaaattgaa cgcgcgaacg
     4021 tctttaaaat gaacggcaaa tggtacctgt tcactgactc ccgcggatca aaaatgacga
     4081 ttgacggcat tacgtctaac gatatttaca tgcttggtta tgtttctaat tctttaactg
     4141 gcccatacaa gccgctgaac aaaactggcc ttgtgttaaa aatggatctt gatcctaacg
     4201 atgtaacctt tacttactca cacttcgctg tacctcaagc gaaaggaaac aatgtcgtga
     4261 ttacaagcta tatgacaaac agaggattct acgcagacaa acaatcaacg tttgcgccaa
     4321 gcttcctgct gaacatcaaa ggcaagaaaa catctgttgt caaagacagc atccttgaac
     4381 aaggacaatt aacagttaac aaataaaaac gcaaaagaaa atgccgatat cctattggca
     4441 ttttctttta tttcttatca acataaaggt gaatcccata tgaactatat aaaagcaggc
     4501 aaatggctaa ccgtattcct aaccttttgg taatgactcc aacttattga tagtgtttta
     4561 tgttcagata atgcccgatg actttgtcat gcagctccac cgattttgag aacgacagcg
     4621 acttccgtcc cagccgtgcc aggtgctgcc tcagattcag gttatgccgc tcaattcgct
     4681 gcgtatatcg cttgctgatt acgtgcagct ttcccttcag gcgggattca tacagcggcc
     4741 agccatccgt catccatatc accacgtcaa agggtgacag caggctcata agacgcccca
     4801 gcgtcgccat agtgcgttca ccgaatacgt gcgcaacaac cgtcttccgg agactgtcat
     4861 acgcgtaaaa cagccagcgc tggcgcgatt tagccccgac atagccccac tgttcgtcca
     4921 tttccgcgca gacgatgacg tcactgcccg gctgtatgcg cgaggttacc gactgcggcc
     4981 tgagtttttt aagtgacgta aaatcgtgtt gaggccaacg cccataatgc gggctgttgc
     5041 ccggcatcca acgccattca tggccatatc aatgattttc tggtgcgtac cgggttgaga
     5101 agcggtgtaa gtgaactgca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5161 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5221 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5281 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5341 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5401 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5461 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     5521 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5581 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5641 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5701 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5761 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     5821 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5881 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5941 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6001 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6061 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6121 aagtttactc atatatactt tagattgatt tatggtgcac tctcagtaca atctgctctg
     6181 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     6241 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     6301 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtca
//
