LOCUS       sequence-484266-        7284 bp    DNA     circular SYN 21-AUG-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7284)
  AUTHORS   Murphy KC, Campellone KG
  TITLE     Lambda Red-mediated recombinogenic engineering of enterohemorrhagic 
            and enteropathogenic E. coli.
  JOURNAL   BMC Mol Biol. 2003 Dec 13;4:11. doi: 10.1186/1471-2199-4-11.
  PUBMED    14672541
REFERENCE   2  (bases 1 to 7284)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 21, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC Mol 
            Biol."; date: "2003-12-13"; volume: "4:11. doi"; pages: " 
            10.1186/1471-2199-4-11"
COMMENT     Sequence Label: pKM201
FEATURES             Location/Qualifiers
     source          1..7284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(84..89,90..105,106..112)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    120..136
                     /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)."
     CDS             169..585
                     /codon_start=1
                     /gene="gam"
                     /product="inhibitor of the host RecBCD nuclease in the 
                     lambda Red system"
                     /label=Gam
                     /note="protects the double-stranded DNA substrate for 
                     recombination"
                     /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
                     IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
                     AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
     CDS             591..1376
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     CDS             1373..2053
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGEDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     primer_bind     complement(2087..2103)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2087..2103)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2140..2160)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2140..2158)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2179..2195)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2179..2195)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2203..2219
                     /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        complement(join(2227..2233,2234..2251,2252..2257))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2272..2293
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2410..2427)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(3765..4715)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      4763..4985
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(5613..6404,6405..6473))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6236..6255
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6474..6578)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6604..7059)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6741..6762)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6953..6972
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7186..7208
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7200..7217
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7201..7217
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7227..7246
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7227..7245
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     join(7270..7284,1..2)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     join(7271..7284,1..3)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tcgataagct tgatatcgaa ttcctgcagc ccgggggatc tccgacatca taacggttct
       61 ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat
      121 tgtgagcgga taacaatttc acacaggaaa cagtcgacgc ttataaaaat ggatattaat
      181 actgaaactg agatcaagca aaagcattca ctaaccccct ttcctgtttt cctaatcagc
      241 ccggcatttc gcgggcgata ttttcacagc tatttcagga gttcagccat gaacgcttat
      301 tacattcagg atcgtcttga ggctcagagc tgggcgcgtc actaccagca gctcgcccgt
      361 gaagagaaag aggcagaact ggcagacgac atggaaaaag gcctgcccca gcacctgttt
      421 gaatcgctat gcatcgatca tttgcaacgc cacggggcca gcaaaaaatc cattacccgt
      481 gcgtttgatg acgatgttga gtttcaggag cgcatggcag aacacatccg gtacatggtt
      541 gaaaccattg ctcaccacca ggttgatatt gattcagagg tataaaacga atgagtactg
      601 cactcgcaac gctggctggg aagctggctg aacgtgtcgg catggattct gtcgacccac
      661 aggaactgat caccactctt cgccagacgg catttaaagg tgatgccagc gatgcgcagt
      721 tcatcgcatt actgatcgtt gccaaccagt acggccttaa tccgtggacg aaagaaattt
      781 acgcctttcc tgataagcag aatggcatcg ttccggtggt gggcgttgat ggctggtccc
      841 gcatcatcaa tgaaaaccag cagtttgatg gcatggactt tgagcaggac aatgaatcct
      901 gtacatgccg gatttaccgc aaggaccgta atcatccgat ctgcgttacc gaatggatgg
      961 atgaatgccg ccgcgaacca ttcaaaactc gcgaaggcag agaaatcacg gggccgtggc
     1021 agtcgcatcc caaacggatg ttacgtcata aagccatgat tcagtgtgcc cgtctggcct
     1081 tcggatttgc tggtatctat gacaaggatg aagccgagcg cattgtcgaa aatactgcat
     1141 acactgcaga acgtcagccg gaacgcgaca tcactccggt taacgatgaa accatgcagg
     1201 agattaacac tctgctgatc gccctggata aaacatggga tgacgactta ttgccgctct
     1261 gttcccagat atttcgccgc gacattcgtg catcgtcaga actgacacag gccgaagcag
     1321 taaaagctct tggattcctg aaacagaaag ccgcagagca gaaggtggca gcatgacacc
     1381 ggacattatc ctgcagcgta ccgggatcga tgtgagagct gtcgaacagg gggaagatgc
     1441 gtggcacaaa ttacggctcg gcgtcatcac cgcttcagaa gttcacaacg tgatagcaaa
     1501 accccgctcc ggaaagaagt ggcctgacat gaaaatgtcc tacttccaca ccctgcttgc
     1561 tgaggtttgc accggtgtgg ctccggaagt taacgctaaa gcactggcct ggggaaaaca
     1621 gtacgagaac gacgccagaa ccctgtttga attcacttcc ggcgtgaatg ttactgaatc
     1681 cccgatcatc tatcgcgacg aaagtatgcg taccgcctgc tctcccgatg gtttatgcag
     1741 tgacggcaac ggccttgaac tgaaatgccc gtttacctcc cgggatttca tgaagttccg
     1801 gctcggtggt ttcgaggcca taaagtcagc ttacatggcc caggtgcagt acagcatgtg
     1861 ggtgacgcga aaaaatgcct ggtactttgc caactatgac ccgcgtatga agcgtgaagg
     1921 cctgcattat gtcgtgattg agcgggatga aaagtacatg gcgagttttg acgagatcgt
     1981 gccggagttc atcgaaaaaa tggacgaggc actggctgaa attggttttg tatttgggga
     2041 gcaatggcga tgacgcatcc tcacgataat atccgggtag gcgcaagatc cactagttct
     2101 agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc
     2161 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     2221 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2281 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2341 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2401 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2461 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2521 catgtgagat ccccacagca agatgcaact ttcgccagat acgacggcgc tctttgccgt
     2581 gctttctgac tttccattcg ccttcaccaa agaccttcag cccggtggaa tcaatcacca
     2641 ggtgtgcgat ttcaccccgg gtggacgttt tgaaactgac attaaccgac tttgcccgct
     2701 tactgacact ggtgtaatcc gggcagcgca acggaacgtt catcagggca aaaatggaat
     2761 caataaaacc ctgcgcagcc cgcagggtca gccggaatac gcgtttaatc accagaacgg
     2821 tggtgatggc gagatcagaa tagcgctggg gccttcctcg tgatgaaggt gttgccgact
     2881 cataccaggc ctgaatcgcc tcatcatcca gccagaaagt gagggagcca cggttgatga
     2941 gagctttgtt gtaggtggac cagttggtga ttctgaactt ttgctttgcc acggaatggt
     3001 ctgtgttgtc gggaggatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc
     3061 aacaaagcca ctttcagtat ggatttgggt gttacggggg ggggttggtg cttaaaatcg
     3121 cgctgaggat ttctgatggt gttaagcggg cggttttgag atgtaaactc gcccgtttaa
     3181 cataatggat cttgcgcgca ccgcccgaac accactcgcc acaaaaaacc gccggaacgt
     3241 ccaaaagtac gggttttgct gcccgcaaac gggctgttct ggtgttgcta gtttgttatc
     3301 agaatcgcag atccggcttc agccggtttg ccggctgaaa gcgctatttc ttccagaatt
     3361 gccatgattt tttccccacg ggaggcgtca ctggctcccg tgttgtcggc agctttgatt
     3421 cgataagcag catcgcctgt ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt
     3481 gtctcaggtg ttcaatttca tgttctagtt gctttgtttt actggtttca cctgttctat
     3541 taggtgttac atgctgttca tctgttacat tgtcgatctg ttcatggtga acagctttaa
     3601 atgcaccaaa aactcgtaaa agctctgatg tatctatctt ttttacaccg ttttcatctg
     3661 tgcatatgga cagttttccc tttgatatgt aacggtgaac agttgttcta cttttgtttg
     3721 ttagtcttga tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat
     3781 ttagccagta tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca
     3841 ttgagatcat acttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga
     3901 atttttgcag ttaaagcatc gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct
     3961 gatgtaatgg ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg
     4021 gttacgagat ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg
     4081 cctcgcttat caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt
     4141 ttcaaaaccc attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg
     4201 aacttaaatt catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt
     4261 tcttttaata accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt
     4321 tccagattat attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta
     4381 aaaactaatt ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaaaatctca
     4441 aagcctttaa ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct
     4501 ttagctaata caccataagc attttcccta ctgatgttca tcatctgaac gtattggtta
     4561 taagtgaacg ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt
     4621 gccacacagc ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct
     4681 agttcatttg ctttgaaaac aactaattca gacatacatc tcaattggtc taggtgattt
     4741 taatcactat accaattgag atgggctagt caatgataat tactagtcct tttcctttga
     4801 gttgtgggta tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa ttctgctaga
     4861 ccctctgtaa attccgctag acctttgtgt gttttttttg tttatattca agtggttata
     4921 atttatagaa taaagaaaga ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat
     4981 aactcactac tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct
     5041 cctctacaaa acagacctta aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc
     5101 aaatcgctga atattccttt tgtctccgac catcaggcac ctgagtcgct gtctttttcg
     5161 tgacattcag ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat ggcactacag
     5221 gcgcctttta tggattcatg caaggaaact acccataata caagaaaagc ccgtcacggg
     5281 cttctcaggg cgttttatgg cgggtctgct atgtggtgct atctgacttt ttgctgttca
     5341 gcagttcctg ccctctgatt ttccagtctg accacttcgg attatcccgt gacaggtcat
     5401 tcagactggc taatgcaccc agtaaggcag cggtatcatc aacaggctta cccgtcttac
     5461 tgtcggggat ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5521 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5581 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5641 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5701 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5761 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5821 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5881 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5941 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccagcg atcaaggcga
     6001 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6061 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6121 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6181 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6241 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6301 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6361 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6421 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6481 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6541 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6601 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     6661 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6721 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6781 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     6841 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6901 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6961 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     7021 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     7081 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     7141 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     7201 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg
     7261 ggccccccct cgaggtcgac ggta
//
