LOCUS       sequence-203476-        5255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains loxP-hyg-cat-loxP cassette for recombineering; CamR, HygR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5255)
  AUTHORS   Murphy KC, Papavinasasundaram K, Sassetti CM
  TITLE     Mycobacterial recombineering.
  JOURNAL   Methods Mol Biol. 2015;1285:177-99. doi: 
            10.1007/978-1-4939-2450-9_10.
  PUBMED    25779316
REFERENCE   2  (bases 1 to 5255)
  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: "Methods Mol
            Biol."; date: "2015"; volume: "1285:177-99. doi"; pages: " 
            10.1007/978-1-4939-2450-9_10"
COMMENT     Sequence Label: pKM343
FEATURES             Location/Qualifiers
     source          1..5255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..20
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4..20
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    22..55
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1173..1275
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1276..1935
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1342..1361)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    1973..2006
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2015..2031)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2016..2032)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(2060..2078)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2099..2115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2099..2115)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2112..2134)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2123..2139
                     /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(2147..2153,2154..2171,2172..2177))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2192..2213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2330..2347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2501..3089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2581..2600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3218..3663
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3664..3750,3751..5085)
                     /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"
     primer_bind     5170..5192
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5184..5201
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5185..5201
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5208..5227
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5208..5226
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cgctctagaa ctagtggatc cataacttcg tataatgtat gctatacgaa gttatactcc
       61 ccgacgtggc cgaccagccc gtcatcgtca acgcctgatc cgcggtgcgg acaggccgtg
      121 tcgcgaccgg ccgtgcggaa ttaagccggc ccgtaccctg tgaatagagg tccgctgtga
      181 cacaagaatc cctgttactt ctcgaccgta ttgattcgga tgattcctac gcgagcctgc
      241 ggaacgacca ggaattctgg gagccgctgg cccgccgagc cctggaggag ctcgggctac
      301 cggtgccgcc ggtgctgcgg gtgcccggcg agagcaccaa ccccgtactg gtcggcgagc
      361 ccggcccggt gatcaagctg ttcggcgagc actggtgcgg tccggagagc ctcgcgtcgg
      421 agtcggaggc gtacgcggtc ctggcggacg ccccggtgcc ggtgccccgc ctcctcggcc
      481 gcggcgagct gcggcccggc accggagcct ggccgtggcc ctacctggtg atgagccgga
      541 tgaccggcac cacctggcgg tccgcaatag acggcacgac cgaccggaac gcgctgctcg
      601 ccctggcccg cgaactcggc cgggtgctcg gccggctgca cagggtgccg ctgaccggga
      661 acaccgtgct caccccccat tccgaggtct tcccggaact gctgcgggaa cgccgcgcgg
      721 cgaccgtcga ggaccaccgc gggtggggct acctctcgcc ccggctgctg gaccgcctgg
      781 aggactggct gccggacgtg gacacgctgc tggccggccg cgaaccccgg ttcgtccacg
      841 gcgacctgca cgggaccaac accttcgtgg acctggccgc gaccgaggtc accgggatcg
      901 tcgacttcac cgacgtctat gcgggagact cccgctacag cctggtgcaa ctgcatctca
      961 acgccttccg gggcgaccgc gagatcctgg ccgcgctgct cgacggggcg cagtggaagc
     1021 ggaccgagga cttcgcccgc gaactgctcg ccttcacctt cctgcacgac ttcgaggtgt
     1081 tcgaggagac cccgctggat ctctccggct tcaccgatcc ggaggaactg gcgcagttcc
     1141 tctgggggcc gccggacacc gcccccggcg cctgatcggc acgtaagagg ttccaacttt
     1201 caccataatg aaataagatc actaccgggc gtattttttg agttatcgag attttcagga
     1261 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
     1321 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
     1381 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
     1441 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg
     1501 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc
     1561 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag
     1621 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct
     1681 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt
     1741 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa
     1801 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtc
     1861 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
     1921 cagggcgggg cgtaacgccc ctctagctgg cgggaacggc aatgctcctg gcataacttc
     1981 gtataatgta tgctatacga agttatacgc ttatcgatac cgtcgacctc gagggggggc
     2041 ccggtaccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg taatcatggt
     2101 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2161 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2221 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2281 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2341 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2401 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2461 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2521 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2581 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2641 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2701 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2761 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2821 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2881 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2941 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3001 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3061 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3121 acgctcagtg catatggagg aatcacttcg caatggccaa gacaattgcg gatccagctg
     3181 caggtcgact ctagaggatc gatccttttt aacccatcac atatacctgc cgttcactat
     3241 tatttagtga aatgagatat tatgatattt tctgaattgt gattaaaaag gcaactttat
     3301 gcccatgcaa cagaaactat aaaaaataca gagaatgaaa agaaacagat agatttttta
     3361 gttctttagg cccgtagtct gcaaatcctt ttatgatttt ctatcaaaca aaagaggaaa
     3421 atagaccagt tgcaatccaa acgagagtct aatagaatga ggtcgaaaag taaatcgcgc
     3481 gggtttgtta ctgataaagc aggcaagacc taaaatgtgt aaagggcaaa gtgtatactt
     3541 tggcgtcacc ccttacatat tttaggtctt tttttattgt gcgtaactaa cttgccatct
     3601 tcaaacagga gggctggaag aagcagaccg ctaacacagt acataaaaaa ggagacatga
     3661 acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat taacctttac taccgcactg
     3721 ctggcaggag gcgcaactca agcgtttgcg aaagaaacga accaaaagcc atataaggaa
     3781 acatacggca tttcccatat tacacgccat gatatgctgc aaatccctga acagcaaaaa
     3841 aatgaaaaat atcaagttcc tgaattcgat tcgtccacaa ttaaaaatat ctcttctgca
     3901 aaaggcctgg acgtttggga cagctggcca ttacaaaacg ctgacggcac tgtcgcaaac
     3961 tatcacggct accacatcgt ctttgcatta gccggagatc ctaaaaatgc ggatgacaca
     4021 tcgatttaca tgttctatca aaaagtcggc gaaacttcta ttgacagctg gaaaaacgct
     4081 ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg attctatcct aaaagaccaa
     4141 acacaagaat ggtcaggttc agccacattt acatctgacg gaaaaatccg tttattctac
     4201 actgatttct ccggtaaaca ttacggcaaa caaacactga caactgcaca agttaacgta
     4261 tcagcatcag acagctcttt gaacatcaac ggtgtagagg attataaatc aatctttgac
     4321 ggtgacggaa aaacgtatca aaatgtacag cagttcatcg atgaaggcaa ctacagctca
     4381 ggcgacaacc atacgctgag agatcctcac tacgtagaag ataaaggcca caaatactta
     4441 gtatttgaag caaacactgg aactgaagat ggctaccaag gcgaagaatc tttatttaac
     4501 aaagcatact atggcaaaag cacatcattc ttccgtcaag aaagtcaaaa acttctgcaa
     4561 agcgataaaa aacgcacggc tgagttagca aacggcgctc tcggtatgat tgagctaaac
     4621 gatgattaca cactgaaaaa agtgatgaaa ccgctgattg catctaacac agtaacagat
     4681 gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat ggtacctgtt cactgactcc
     4741 cgcggatcaa aaatgacgat tgacggcatt acgtctaacg atatttacat gcttggttat
     4801 gtttctaatt ctttaactgg cccatacaag ccgctgaaca aaactggcct tgtgttaaaa
     4861 atggatcttg atcctaacga tgtaaccttt acttactcac acttcgctgt acctcaagcg
     4921 aaaggaaaca atgtcgtgat tacaagctat atgacaaaca gaggattcta cgcagacaaa
     4981 caatcaacgt ttgcgccaag cttcctgctg aacatcaaag gcaagaaaac atctgttgtc
     5041 aaagacagca tccttgaaca aggacaatta acagttaaca aataaaaacg caaaagaaaa
     5101 tgccgatatc ctattggctt aattaaggat gtgctgcaag gcgattaagt tgggtaacgc
     5161 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac
     5221 tatagggcga attggagctc caccgcggtg gcggc
//
