LOCUS       sequence-220076-        7643 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 7643)
  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 7643)
  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: "BMC Mol 
            Biol."; date: "2003-12-13"; volume: "4:11. doi"; pages: " 
            10.1186/1471-2199-4-11"
COMMENT     Sequence Label: pKM200
FEATURES             Location/Qualifiers
     source          1..7643
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    359..380
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(395..400,401..418,419..425)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    433..449
                     /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)."
     primer_bind     457..473
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     457..473
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        join(564..569,570..585,586..592)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    600..616
                     /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             649..1065
                     /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             1071..1856
                     /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             1853..2533
                     /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(2593..2610)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2593..2609)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2602..2624)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(3554..4213)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4128..4147
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4214..4316)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4433..4451)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4519..4623
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4842..4861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(6107..7057)
                     /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      7105..7327
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 gggtctgcta tgtggtgcta tctgactttt tgctgttcag cagttcctgc cctctgattt
       61 tccagtctga ccacttcgga ttatcccgtg acaggtcatt cagactggct aatgcaccca
      121 gtaaggcagc ggtatcatca acaggcttac ccgtcttact gtcggggatc gacgctctcc
      181 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
      241 cgccgcaagg aatggtgcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
      301 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
      361 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      421 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
      481 acgccaagct tgcatgcctg caggtcgact ctagaggatc tccgacatca taacggttct
      541 ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat
      601 tgtgagcgga taacaatttc acacaggaaa cagtcgacgc ttataaaaat ggatattaat
      661 actgaaactg agatcaagca aaagcattca ctaaccccct ttcctgtttt cctaatcagc
      721 ccggcatttc gcgggcgata ttttcacagc tatttcagga gttcagccat gaacgcttat
      781 tacattcagg atcgtcttga ggctcagagc tgggcgcgtc actaccagca gctcgcccgt
      841 gaagagaaag aggcagaact ggcagacgac atggaaaaag gcctgcccca gcacctgttt
      901 gaatcgctat gcatcgatca tttgcaacgc cacggggcca gcaaaaaatc cattacccgt
      961 gcgtttgatg acgatgttga gtttcaggag cgcatggcag aacacatccg gtacatggtt
     1021 gaaaccattg ctcaccacca ggttgatatt gattcagagg tataaaacga atgagtactg
     1081 cactcgcaac gctggctggg aagctggctg aacgtgtcgg catggattct gtcgacccac
     1141 aggaactgat caccactctt cgccagacgg catttaaagg tgatgccagc gatgcgcagt
     1201 tcatcgcatt actgatcgtt gccaaccagt acggccttaa tccgtggacg aaagaaattt
     1261 acgcctttcc tgataagcag aatggcatcg ttccggtggt gggcgttgat ggctggtccc
     1321 gcatcatcaa tgaaaaccag cagtttgatg gcatggactt tgagcaggac aatgaatcct
     1381 gtacatgccg gatttaccgc aaggaccgta atcatccgat ctgcgttacc gaatggatgg
     1441 atgaatgccg ccgcgaacca ttcaaaactc gcgaaggcag agaaatcacg gggccgtggc
     1501 agtcgcatcc caaacggatg ttacgtcata aagccatgat tcagtgtgcc cgtctggcct
     1561 tcggatttgc tggtatctat gacaaggatg aagccgagcg cattgtcgaa aatactgcat
     1621 acactgcaga acgtcagccg gaacgcgaca tcactccggt taacgatgaa accatgcagg
     1681 agattaacac tctgctgatc gccctggata aaacatggga tgacgactta ttgccgctct
     1741 gttcccagat atttcgccgc gacattcgtg catcgtcaga actgacacag gccgaagcag
     1801 taaaagctct tggattcctg aaacagaaag ccgcagagca gaaggtggca gcatgacacc
     1861 ggacattatc ctgcagcgta ccgggatcga tgtgagagct gtcgaacagg gggaagatgc
     1921 gtggcacaaa ttacggctcg gcgtcatcac cgcttcagaa gttcacaacg tgatagcaaa
     1981 accccgctcc ggaaagaagt ggcctgacat gaaaatgtcc tacttccaca ccctgcttgc
     2041 tgaggtttgc accggtgtgg ctccggaagt taacgctaaa gcactggcct ggggaaaaca
     2101 gtacgagaac gacgccagaa ccctgtttga attcacttcc ggcgtgaatg ttactgaatc
     2161 cccgatcatc tatcgcgacg aaagtatgcg taccgcctgc tctcccgatg gtttatgcag
     2221 tgacggcaac ggccttgaac tgaaatgccc gtttacctcc cgggatttca tgaagttccg
     2281 gctcggtggt ttcgaggcca taaagtcagc ttacatggcc caggtgcagt acagcatgtg
     2341 ggtgacgcga aaaaatgcct ggtactttgc caactatgac ccgcgtatga agcgtgaagg
     2401 cctgcattat gtcgtgattg agcgggatga aaagtacatg gcgagttttg acgagatcgt
     2461 gccggagttc atcgaaaaaa tggacgaggc actggctgaa attggttttg tatttgggga
     2521 gcaatggcga tgacgcatcc tcacgataat atccgggtag gcgcaagatc cccgggtacc
     2581 gagctcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2641 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2701 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgag cttatcgatg
     2761 ataagctgtc aaacatggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     2821 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     2881 catggcggcc gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag gctggatggc
     2941 cttccccatt atgattcttc tcgcttccgg cggcatcggg atgcccgcgt tgcaggccat
     3001 gctgtccagg caggtagatg acgaccatca gggacagctt caaggatcgc tcgcggctct
     3061 taccagccta acttcgatca ttggaccgct gatcgtcacg gcgatttatg ccgcctcggc
     3121 gagcacatgg aacgggttgg catggattgt aggcgccgcc ctataccttg tctgcctccc
     3181 cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac
     3241 ctcgctaacg gattcaccgt ttttatcagg ctctgggagg cagaataaat gatcatatcg
     3301 tcaattatta cctccacggg gagagcctga gcaaactggc ctcaggcatt tgagaagcac
     3361 acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct
     3421 atttaacgac cctgccctga accgacgacc gggtcgaatt tgctttcgaa tttctgccat
     3481 tcatccgctt attatcactt attcaggcgt agcaaccagg cgtttaaggg caccaataac
     3541 tgccttaaaa aaattacgcc ccgccctgcc actcatcgca gtactgttgt aattcattaa
     3601 gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca
     3661 tcagcacctt gtcgccttgc gtataatatt tgcccatggt gaaaacgggg gcgaagaagt
     3721 tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga
     3781 cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg
     3841 ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga
     3901 gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc
     3961 atatcaccag ctcaccgtct ttcattgcca tacggaattc cggatgagca ttcatcaggc
     4021 gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta
     4081 aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa
     4141 atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga
     4201 tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc
     4261 ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt
     4321 ctcattttcg ccaaaagttg gcccagggct tcccggtatc aacagggaca ccaggattta
     4381 tttattctgc gaagtgatct tccgtcacag gtatttattc gaagacgaaa gggcctcgtg
     4441 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4501 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4561 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4621 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4681 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4741 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4801 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4861 tcccgtgttg atccccacag caagatgcaa ctttcgccag atacgacggc gctctttgcc
     4921 gtgctttctg actttccatt cgccttcacc aaagaccttc agcccggtgg aatcaatcac
     4981 caggtgtgcg atttcacccc gggtggacgt tttgaaactg acattaaccg actttgcccg
     5041 cttactgaca ctggtgtaat ccgggcagcg caacggaacg ttcatcaggg caaaaatgga
     5101 atcaataaaa ccctgcgcag cccgcagggt cagccggaat acgcgtttaa tcaccagaac
     5161 ggtggtgatg gcgagatcag aatagcgctg gggccttcct cgtgatgaag gtgttgccga
     5221 ctcataccag gcctgaatcg cctcatcatc cagccagaaa gtgagggagc cacggttgat
     5281 gagagctttg ttgtaggtgg accagttggt gattctgaac ttttgctttg ccacggaatg
     5341 gtctgtgttg tcgggaggat gcgtgatctg atccttcaac tcagcaaaag ttcgatttat
     5401 tcaacaaagc cactttcagt atggatttgg gtgttacggg ggggggttgg tgcttaaaat
     5461 cgcgctgagg atttctgatg gtgttaagcg ggcggttttg agatgtaaac tcgcccgttt
     5521 aacataatgg atcttgcgcg caccgcccga acaccactcg ccacaaaaaa ccgccggaac
     5581 gtccaaaagt acgggttttg ctgcccgcaa acgggctgtt ctggtgttgc tagtttgtta
     5641 tcagaatcgc agatccggct tcagccggtt tgccggctga aagcgctatt tcttccagaa
     5701 ttgccatgat tttttcccca cgggaggcgt cactggctcc cgtgttgtcg gcagctttga
     5761 ttcgataagc agcatcgcct gtttcaggct gtctatgtgt gactgttgag ctgtaacaag
     5821 ttgtctcagg tgttcaattt catgttctag ttgctttgtt ttactggttt cacctgttct
     5881 attaggtgtt acatgctgtt catctgttac attgtcgatc tgttcatggt gaacagcttt
     5941 aaatgcacca aaaactcgta aaagctctga tgtatctatc ttttttacac cgttttcatc
     6001 tgtgcatatg gacagttttc cctttgatat gtaacggtga acagttgttc tacttttgtt
     6061 tgttagtctt gatgcttcac tgatagatac aagagccata agaacctcag atccttccgt
     6121 atttagccag tatgttctct agtgtggttc gttgtttttg cgtgagccat gagaacgaac
     6181 cattgagatc atacttactt tgcatgtcac tcaaaaattt tgcctcaaaa ctggtgagct
     6241 gaatttttgc agttaaagca tcgtgtagtg tttttcttag tccgttatgt aggtaggaat
     6301 ctgatgtaat ggttgttggt attttgtcac cattcatttt tatctggttg ttctcaagtt
     6361 cggttacgag atccatttgt ctatctagtt caacttggaa aatcaacgta tcagtcgggc
     6421 ggcctcgctt atcaaccacc aatttcatat tgctgtaagt gtttaaatct ttacttattg
     6481 gtttcaaaac ccattggtta agccttttaa actcatggta gttattttca agcattaaca
     6541 tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt gtgagttttc ttttgtgtta
     6601 gttcttttaa taaccactca taaatcctca tagagtattt gttttcaaaa gacttaacat
     6661 gttccagatt atattttatg aattttttta actggaaaag ataaggcaat atctcttcac
     6721 taaaaactaa ttctaatttt tcgcttgaga acttggcata gtttgtccac tggaaaatct
     6781 caaagccttt aaccaaagga ttcctgattt ccacagttct cgtcatcagc tctctggttg
     6841 ctttagctaa tacaccataa gcattttccc tactgatgtt catcatctga acgtattggt
     6901 tataagtgaa cgataccgtc cgttctttcc ttgtagggtt ttcaatcgtg gggttgagta
     6961 gtgccacaca gcataaaatt agcttggttt catgctccgt taagtcatag cgactaatcg
     7021 ctagttcatt tgctttgaaa acaactaatt cagacataca tctcaattgg tctaggtgat
     7081 tttaatcact ataccaattg agatgggcta gtcaatgata attactagtc cttttccttt
     7141 gagttgtggg tatctgtaaa ttctgctaga cctttgctgg aaaacttgta aattctgcta
     7201 gaccctctgt aaattccgct agacctttgt gtgttttttt tgtttatatt caagtggtta
     7261 taatttatag aataaagaaa gaataaaaaa agataaaaag aatagatccc agccctgtgt
     7321 ataactcact actttagtca gttccgcagt attacaaaag gatgtcgcaa acgctgtttg
     7381 ctcctctaca aaacagacct taaaacccta aaggcttaag tagcaccctc gcaagctcgg
     7441 gcaaatcgct gaatattcct tttgtctccg accatcaggc acctgagtcg ctgtcttttt
     7501 cgtgacattc agttcgctgc gctcacggct ctggcagtga atgggggtaa atggcactac
     7561 aggcgccttt tatggattca tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg
     7621 ggcttctcag ggcgttttat ggc
//
