LOCUS       sequence-275822-        6608 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  loxP (empty) expression cassette: n-terminal GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6608)
  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 6608)
  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: pGL2316
FEATURES             Location/Qualifiers
     source          1..6608
                     /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             join(902..904,905..907,908..1618)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(902..904,905..907,908..1618)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(947..968)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1208..1227)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1555..1576
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1637..1717
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             1664..1744
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             1691..1771
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             1718..1798
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     protein_bind    complement(1808..1841)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2547..3146
                     /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(2547..2566)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3043..3063
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4161..4178
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(4733..4832)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4849..4868)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4850..4868)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4873..4889)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4873..4889)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4991..5010)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5002..5811
                     /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(5069..5088)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5958..6546
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6447..6466
                     /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 gatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      961 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1021 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1081 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1141 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1201 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1261 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1321 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1381 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1441 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1501 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1561 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg
     1621 taccatcgat actagttacc cttacgatgt gcctgattac gcgtacccat acgacgtgcc
     1681 agactacgca tacccgtacg atgtgcccga ttacgcatac ccttacgatg tgcctgatta
     1741 cgcgtaccca tacgacgtgc cagactacgc atacccgtac gatgtgcccg attacgcatg
     1801 atctagaata acttcgtata gcatacatta tacgaagtta tggctgcagc gcgtggatgt
     1861 cgcgcagcgc aagaccccgt gcgcctgaaa cgtgaaggga ggtcgagaag cgtgccgcat
     1921 gaggccttaa ggcaggaaag tgaaaagagc tcgacgcgag tgcagcacgc gcgcatcgac
     1981 aggacggaat agacactcag ccccttcccc tttcaaaaac tgaatggacg gacatcgtaa
     2041 cgcgcttctc tcccctccac tccccctgtc cctgtctcct tgacgtgtgc tgctctcgtg
     2101 agcgacagat gaagcgaggc gtagaaggaa aggggggggg agcaggcctg cacacacaca
     2161 cacacacaca cacacacaca cacacacaca cacacacaca cacgaaaagc aagtttgagg
     2221 cagacgtggt ttcacacgat tcctgtgcaa gatgcgtgcc tgcgtgacgg gtgtggctgt
     2281 gtatgcatgc gtgaagggtt accgcaacga tgcgcaatgg actcccccgc tttccatttc
     2341 gtcaccttcc gcctctctct ctctctctct caccatctac gcgtgcacat catcaactgt
     2401 ctcttgtcgg tgctcaccac cctcaaccac ccctcacttt caaggcttcc cgaacgcaca
     2461 caaaaggcgt gaaaaccgct cgcggtgtgt tgagccgtcc accgtgccct ccccctgtcc
     2521 ccgggggatc cactagacga ccttccatga ccgagtacaa gcccacggtg cgcctcgcca
     2581 cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     2641 ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     2701 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     2761 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     2821 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     2881 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     2941 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     3001 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     3061 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3121 gcatgacccg caagcccggt gcctaggcta gcgctctcga gaattgcccg ctttccattt
     3181 cgtcaccttc cgcctctctc tctctctctc tcaccatcta cgcgtgcaca tcatcaactg
     3241 tctcttgtcg gtgctcacca ccctcaacca cccctcactt tcaaggcttc ccgaacgcac
     3301 acaaaaggcg tgaaaaccgc tcgcggtgtg ttgagccgtc caccgtagct gatcctatct
     3361 atctcccccg cttatacacc atgggaagct cttgcacgaa ggactccgca aaggagcccc
     3421 agaaaagtgc ggatggatct atggaggatc caatggttag taaaggagaa gaaaataaca
     3481 tggcaatcat taaggagttc atgagattca aagttcacat ggaaggttct gtaaatggac
     3541 atgaatttga aatagaaggt gaaggagaag gaaggcctta tgaaggaacc caaaccgcga
     3601 agctaaaagt tactaagggt ggcccattac catttgcatg ggatatcctt agccctcaat
     3661 tcatgtatgg gtcaaaggct tatgtcaagc accccgccga cattccagac tatctaaagt
     3721 tatcttttcc cgaagggttt aagtgggagc gtgtgatgaa cttcgaagac ggtggcgtgg
     3781 taacagtgac tcaggattcg tccctgcaag atggtgaatt tatctacaaa gtcaaattaa
     3841 gaggaactaa ctttccatct ggcggcccgg ttatgcaaaa aaagacaatg ggctgggagg
     3901 cctcctcaga acgaatgtac cctgaagatg gtgccttgaa gggtgagatt aaacaaagat
     3961 tgaaattgaa agatggtgga cattatgacg ctgaggttaa aacgacatac aaagctaaga
     4021 aacctgtcca gctcccaggt gcttacaatg taaatataaa acttgatatt acatcacata
     4081 atgaagatta tacgatagtt gaacaatacg aaagggctga ggggagacat agtactggtg
     4141 gcatggatga actatacaaa catcatcatc atcatcattg aggatcccgc ggccgctaga
     4201 tgtgtgccct tgtgtgcgtg tgtgtttggt ggtttgcagc gatgcccggc gcgtgtgggc
     4261 acctccttgg gtgcgcgccc gccgtggcag ctgcgcgtgc gtgcgagatg tgaggcagag
     4321 gaagaggaag gcgatgcggg cgacagcgca gcgaggtgcg gcggagcgta ggggggaaat
     4381 ggacgagcag gcgcgctgtg aatcggagct gcggcaccac ccaagtcgtg gtgccccgcg
     4441 aatggctgtt ctgcccgccc tcgcttcacg cctccccctc ccctcgcgtg ccctcgcgtg
     4501 gcctcccttg ttatccctct ctctcgcacg cacacgcaca cgcgtatacg cgagcccgct
     4561 attctgcctt cgtctggctc tttgtatttt gcttgcttct tcagcacact tgtgtgctgt
     4621 gcgttcagcg atatcttcca ctactttgtt ttctcctccc cctcgggagg tgcttcgctt
     4681 gtgctttgac ggtggtgcgt ggctgctggg ttatgtgccg ggcgcccatg gaacccagct
     4741 ttcttgtaca aagttggcat tataagaaag cattgcttat caatttgttg caacgaacag
     4801 gtcactatca gtcaaaataa aatcattatt tgccatccag ctgatatccc ctatagtgag
     4861 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
     4921 tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata
     4981 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta
     5041 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     5101 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa
     5161 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     5221 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     5281 ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat
     5341 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     5401 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     5461 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     5521 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     5581 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     5641 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     5701 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     5761 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg
     5821 gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca
     5881 tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa
     5941 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6001 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6061 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6121 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6181 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6241 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6301 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6361 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6421 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6481 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6541 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6601 cacatgtt
//
