LOCUS       sequence-275819-        5907 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  loxP (empty) expression cassette: c-terminal 6xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5907)
  AUTHORS   Duncan SM, Myburgh E, Alves-Ferreira EV, Mottram JC
  TITLE     DiCre-Based Inducible Disruption of Leishmania Genes.
  JOURNAL   Methods Mol Biol. 2019;1971:211-224. doi: 
            10.1007/978-1-4939-9210-2_10.
  PUBMED    30980305
REFERENCE   2  (bases 1 to 5907)
  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: "2019"; volume: "1971:211-224. doi"; pages: " 
            10.1007/978-1-4939-9210-2_10"
COMMENT     Sequence Label: pGL2314
FEATURES             Location/Qualifiers
     source          1..5907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(675..708)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             932..1012
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             959..1039
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             986..1066
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             1013..1093
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     protein_bind    complement(1103..1136)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1846..2445
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1846..1865)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2342..2362
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3460..3477
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(4032..4131)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4148..4167)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4149..4167)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4172..4188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4172..4188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4290..4309)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4301..5110
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4368..4387)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5257..5845
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5746..5765
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctta gatcataact tcgtatagca tacattatac gaagttataa ttgcccgctt
      721 tccatttcgt caccttccgc ctctctctct ctctctctca ccatctacgc gtgcacatca
      781 tcaactgtct cttgtcggtg ctcaccaccc tcaaccaccc ctcactttca aggcttcccg
      841 aacgcacaca aaaggcgtga aaaccgctcg cgtgtgttga gccgtccacc gtagccatat
      901 ggaattcagt actggtacca tcgatactag ttacccttac gatgtgcctg attacgcgta
      961 cccatacgac gtgccagact acgcataccc gtacgatgtg cccgattacg cataccctta
     1021 cgatgtgcct gattacgcgt acccatacga cgtgccagac tacgcatacc cgtacgatgt
     1081 gcccgattac gcatgatcta gaataacttc gtatagcata cattatacga agttatggct
     1141 gcagcgcgtg gatgtcgcgc agcgcaagac cccgtgcgcc tgaaacgtga agggaggtcg
     1201 agaagcgtgc cgcatgaggc cttaaggcag gaaagtgaaa agagctcgac gcgagtgcag
     1261 cacgcgcgca tcgacaggac ggaatagaca ctcagcccct tcccctttca aaaactgaat
     1321 ggacggacat cgtaacgcgc ttctctcccc tccactcccc ctgtccctgt ctccttgacg
     1381 tgtgctgctc tcgtgagcga cagatgaagc gaggcgtaga aggaaagggg gggggagcag
     1441 gcctgcacac acacacacac acacacacac acacacacac acacacacac acacacacac
     1501 acgaaaagca agtttgaggc agacgtggtt tcacacgatt cctgtgcaag atgcgtgcct
     1561 gcgtgacggg tgtggctgtg tatgcatgcg tgaagggtta ccgcaacgat gcgcaatgga
     1621 ctcccccgct ttccatttcg tcaccttccg cctctctctc tctctctctc accatctacg
     1681 cgtgcacatc atcaactgtc tcttgtcggt gctcaccacc ctcaaccacc cctcactttc
     1741 aaggcttccc gaacgcacac aaaaggcgtg aaaaccgctc gcggtgtgtt gagccgtcca
     1801 ccgtgccctc cccctgtccc cgggggatcc actagacgac cttccatgac cgagtacaag
     1861 cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc
     1921 gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca catcgagcgg
     1981 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     2041 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     2101 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     2161 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     2221 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     2281 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     2341 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     2401 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctaggctag cgctctcgag
     2461 aattgcccgc tttccatttc gtcaccttcc gcctctctct ctctctctct caccatctac
     2521 gcgtgcacat catcaactgt ctcttgtcgg tgctcaccac cctcaaccac ccctcacttt
     2581 caaggcttcc cgaacgcaca caaaaggcgt gaaaaccgct cgcggtgtgt tgagccgtcc
     2641 accgtagctg atcctatcta tctcccccgc ttatacacca tgggaagctc ttgcacgaag
     2701 gactccgcaa aggagcccca gaaaagtgcg gatggatcta tggaggatcc aatggttagt
     2761 aaaggagaag aaaataacat ggcaatcatt aaggagttca tgagattcaa agttcacatg
     2821 gaaggttctg taaatggaca tgaatttgaa atagaaggtg aaggagaagg aaggccttat
     2881 gaaggaaccc aaaccgcgaa gctaaaagtt actaagggtg gcccattacc atttgcatgg
     2941 gatatcctta gccctcaatt catgtatggg tcaaaggctt atgtcaagca ccccgccgac
     3001 attccagact atctaaagtt atcttttccc gaagggttta agtgggagcg tgtgatgaac
     3061 ttcgaagacg gtggcgtggt aacagtgact caggattcgt ccctgcaaga tggtgaattt
     3121 atctacaaag tcaaattaag aggaactaac tttccatctg gcggcccggt tatgcaaaaa
     3181 aagacaatgg gctgggaggc ctcctcagaa cgaatgtacc ctgaagatgg tgccttgaag
     3241 ggtgagatta aacaaagatt gaaattgaaa gatggtggac attatgacgc tgaggttaaa
     3301 acgacataca aagctaagaa acctgtccag ctcccaggtg cttacaatgt aaatataaaa
     3361 cttgatatta catcacataa tgaagattat acgatagttg aacaatacga aagggctgag
     3421 gggagacata gtactggtgg catggatgaa ctatacaaac atcatcatca tcatcattga
     3481 ggatcccgcg gccgctagat gtgtgccctt gtgtgcgtgt gtgtttggtg gtttgcagcg
     3541 atgcccggcg cgtgtgggca cctccttggg tgcgcgcccg ccgtggcagc tgcgcgtgcg
     3601 tgcgagatgt gaggcagagg aagaggaagg cgatgcgggc gacagcgcag cgaggtgcgg
     3661 cggagcgtag gggggaaatg gacgagcagg cgcgctgtga atcggagctg cggcaccacc
     3721 caagtcgtgg tgccccgcga atggctgttc tgcccgccct cgcttcacgc ctccccctcc
     3781 cctcgcgtgc cctcgcgtgg cctcccttgt tatccctctc tctcgcacgc acacgcacac
     3841 gcgtatacgc gagcccgcta ttctgccttc gtctggctct ttgtattttg cttgcttctt
     3901 cagcacactt gtgtgctgtg cgttcagcga tatcttccac tactttgttt tctcctcccc
     3961 ctcgggaggt gcttcgcttg tgctttgacg gtggtgcgtg gctgctgggt tatgtgccgg
     4021 gcgcccatgg aacccagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc
     4081 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
     4141 tgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc
     4201 cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     4261 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     4321 aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     4381 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     4441 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     4501 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     4561 tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt
     4621 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     4681 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     4741 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     4801 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     4861 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     4921 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     4981 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     5041 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     5101 gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg
     5161 acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     5221 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5281 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5341 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5401 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5461 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5521 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5581 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5641 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5701 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5761 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5821 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5881 cttttgctgg ccttttgctc acatgtt
//
