LOCUS       sequence-275821-        6458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  loxP (empty) expression cassette: c-terminal GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6458)
  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 6458)
  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: pGL2315
FEATURES             Location/Qualifiers
     source          1..6458
                     /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(932..934,935..937,938..1651)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(932..934,935..937,938..1648)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(977..998)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1238..1257)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1585..1606
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1658..1691)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2397..2996
                     /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(2397..2416)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2893..2913
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4011..4028
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(4583..4682)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4699..4718)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4700..4718)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4723..4739)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4723..4739)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4841..4860)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4852..5661
                     /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(4919..4938)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5808..6396
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6297..6316
                     /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 tatggtgagc aagggcgagg agctgttcac
      961 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1021 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1081 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1141 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1201 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1261 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1321 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1381 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1441 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1501 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     1561 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1621 cactctcggc atggacgagc tgtacaagtg atctagaata acttcgtata gcatacatta
     1681 tacgaagtta tggctgcagc gcgtggatgt cgcgcagcgc aagaccccgt gcgcctgaaa
     1741 cgtgaaggga ggtcgagaag cgtgccgcat gaggccttaa ggcaggaaag tgaaaagagc
     1801 tcgacgcgag tgcagcacgc gcgcatcgac aggacggaat agacactcag ccccttcccc
     1861 tttcaaaaac tgaatggacg gacatcgtaa cgcgcttctc tcccctccac tccccctgtc
     1921 cctgtctcct tgacgtgtgc tgctctcgtg agcgacagat gaagcgaggc gtagaaggaa
     1981 aggggggggg agcaggcctg cacacacaca cacacacaca cacacacaca cacacacaca
     2041 cacacacaca cacgaaaagc aagtttgagg cagacgtggt ttcacacgat tcctgtgcaa
     2101 gatgcgtgcc tgcgtgacgg gtgtggctgt gtatgcatgc gtgaagggtt accgcaacga
     2161 tgcgcaatgg actcccccgc tttccatttc gtcaccttcc gcctctctct ctctctctct
     2221 caccatctac gcgtgcacat catcaactgt ctcttgtcgg tgctcaccac cctcaaccac
     2281 ccctcacttt caaggcttcc cgaacgcaca caaaaggcgt gaaaaccgct cgcggtgtgt
     2341 tgagccgtcc accgtgccct ccccctgtcc ccgggggatc cactagacga ccttccatga
     2401 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca
     2461 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc
     2521 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     2581 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     2641 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     2701 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     2761 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     2821 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     2881 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     2941 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctaggcta
     3001 gcgctctcga gaattgcccg ctttccattt cgtcaccttc cgcctctctc tctctctctc
     3061 tcaccatcta cgcgtgcaca tcatcaactg tctcttgtcg gtgctcacca ccctcaacca
     3121 cccctcactt tcaaggcttc ccgaacgcac acaaaaggcg tgaaaaccgc tcgcggtgtg
     3181 ttgagccgtc caccgtagct gatcctatct atctcccccg cttatacacc atgggaagct
     3241 cttgcacgaa ggactccgca aaggagcccc agaaaagtgc ggatggatct atggaggatc
     3301 caatggttag taaaggagaa gaaaataaca tggcaatcat taaggagttc atgagattca
     3361 aagttcacat ggaaggttct gtaaatggac atgaatttga aatagaaggt gaaggagaag
     3421 gaaggcctta tgaaggaacc caaaccgcga agctaaaagt tactaagggt ggcccattac
     3481 catttgcatg ggatatcctt agccctcaat tcatgtatgg gtcaaaggct tatgtcaagc
     3541 accccgccga cattccagac tatctaaagt tatcttttcc cgaagggttt aagtgggagc
     3601 gtgtgatgaa cttcgaagac ggtggcgtgg taacagtgac tcaggattcg tccctgcaag
     3661 atggtgaatt tatctacaaa gtcaaattaa gaggaactaa ctttccatct ggcggcccgg
     3721 ttatgcaaaa aaagacaatg ggctgggagg cctcctcaga acgaatgtac cctgaagatg
     3781 gtgccttgaa gggtgagatt aaacaaagat tgaaattgaa agatggtgga cattatgacg
     3841 ctgaggttaa aacgacatac aaagctaaga aacctgtcca gctcccaggt gcttacaatg
     3901 taaatataaa acttgatatt acatcacata atgaagatta tacgatagtt gaacaatacg
     3961 aaagggctga ggggagacat agtactggtg gcatggatga actatacaaa catcatcatc
     4021 atcatcattg aggatcccgc ggccgctaga tgtgtgccct tgtgtgcgtg tgtgtttggt
     4081 ggtttgcagc gatgcccggc gcgtgtgggc acctccttgg gtgcgcgccc gccgtggcag
     4141 ctgcgcgtgc gtgcgagatg tgaggcagag gaagaggaag gcgatgcggg cgacagcgca
     4201 gcgaggtgcg gcggagcgta ggggggaaat ggacgagcag gcgcgctgtg aatcggagct
     4261 gcggcaccac ccaagtcgtg gtgccccgcg aatggctgtt ctgcccgccc tcgcttcacg
     4321 cctccccctc ccctcgcgtg ccctcgcgtg gcctcccttg ttatccctct ctctcgcacg
     4381 cacacgcaca cgcgtatacg cgagcccgct attctgcctt cgtctggctc tttgtatttt
     4441 gcttgcttct tcagcacact tgtgtgctgt gcgttcagcg atatcttcca ctactttgtt
     4501 ttctcctccc cctcgggagg tgcttcgctt gtgctttgac ggtggtgcgt ggctgctggg
     4561 ttatgtgccg ggcgcccatg gaacccagct ttcttgtaca aagttggcat tataagaaag
     4621 cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt
     4681 tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg
     4741 cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
     4801 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     4861 attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg
     4921 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg
     4981 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     5041 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     5101 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca
     5161 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     5221 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     5281 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     5341 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     5401 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     5461 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     5521 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     5581 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     5641 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt
     5701 acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg
     5761 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5821 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5881 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5941 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6001 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6061 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6121 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6181 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6241 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6301 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6361 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6421 cggttcctgg ccttttgctg gccttttgct cacatgtt
//
