LOCUS       sequence-291824-        4036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains loxP-hyg-loxP cassette for recombineering; HygR, AmpR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4036)
  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 4036)
  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: pKM342
FEATURES             Location/Qualifiers
     source          1..4036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     473..489
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     473..489
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    491..524
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1680..1713
                     /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(1815..1831)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1815..1831)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1828..1850)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1839..1855
                     /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(1863..1869,1870..1887,1888..1893))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1908..1929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2046..2063)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2217..2805)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2297..2316)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2976..3767,3768..3836))
                     /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     3599..3618
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3837..3941)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4009..4027
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt acttaagaga tctactagtg
      421 gcgcgccgac tcactatagg gcgaattgga gctccaccgc ggtggcggcc gctctagaac
      481 tagtggatcc ataacttcgt ataatgtatg ctatacgaag ttatactccc cgacgtggcc
      541 gaccagcccg tcatcgtcaa cgcctgatcc gcggtgcgga caggccgtgt cgcgaccggc
      601 cgtgcggaat taagccggcc cgtaccctgt gaatagaggt ccgctgtgac acaagaatcc
      661 ctgttacttc tcgaccgtat tgattcggat gattcctacg cgagcctgcg gaacgaccag
      721 gaattctggg agccgctggc ccgccgagcc ctggaggagc tcgggctacc ggtgccgccg
      781 gtgctgcggg tgcccggcga gagcaccaac cccgtactgg tcggcgagcc cggcccggtg
      841 atcaagctgt tcggcgagca ctggtgcggt ccggagagcc tcgcgtcgga gtcggaggcg
      901 tacgcggtcc tggcggacgc cccggtgccg gtgccccgcc tcctcggccg cggcgagctg
      961 cggcccggca ccggagcctg gccgtggccc tacctggtga tgagccggat gaccggcacc
     1021 acctggcggt ccgcaataga cggcacgacc gaccggaacg cgctgctcgc cctggcccgc
     1081 gaactcggcc gggtgctcgg ccggctgcac agggtgccgc tgaccgggaa caccgtgctc
     1141 accccccatt ccgaggtctt cccggaactg ctgcgggaac gccgcgcggc gaccgtcgag
     1201 gaccaccgcg ggtggggcta cctctcgccc cggctgctgg accgcctgga ggactggctg
     1261 ccggacgtgg acacgctgct ggccggccgc gaaccccggt tcgtccacgg cgacctgcac
     1321 gggaccaaca ccttcgtgga cctggccgcg accgaggtca ccgggatcgt cgacttcacc
     1381 gacgtctatg cgggagactc ccgctacagc ctggtgcaac tgcatctcaa cgccttccgg
     1441 ggcgaccgcg agatcctggc cgcgctgctc gacggggcgc agtggaagcg gaccgaggac
     1501 ttcgcccgcg aactgctcgc cttcaccttc ctgcacgact tcgaggtgtt cgaggagacc
     1561 ccgctggatc tctccggctt caccgatccg gaggaactgg cgcagttcct ctgggggccg
     1621 ccggacaccg cccccggcgc ctgatcggca cgtaagaggt tccaactttc accataatga
     1681 taacttcgta taatgtatgc tatacgaagt tatggcgcgc cggtaccctc gagtctagaa
     1741 gtacttaatt cgagctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc
     1801 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1861 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1921 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1981 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2041 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2101 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2161 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2221 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2281 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2341 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2401 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2461 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2521 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2581 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2641 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2701 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2761 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2821 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2881 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2941 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3001 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3061 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3121 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3181 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3241 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3301 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3361 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3421 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3481 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3541 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3601 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3661 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3721 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3781 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3841 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3901 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3961 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     4021 acgaggccct ttcgtc
//
