LOCUS       sequence-275799-        6732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous Locus DiCre Expression Cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6732)
  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 6732)
  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: pGL2313
FEATURES             Location/Qualifiers
     source          1..6732
                     /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"
     CDS             880..900
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             907..1227
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             2431..2451
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2458..2733
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRIS"
     CDS             4144..4542
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(4666..4677)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(4857..4956)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4973..4992)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4974..4992)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4997..5013)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4997..5013)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5115..5134)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5126..5935
                     /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(5193..5212)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6082..6670
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6571..6590
                     /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 agcaggcttg ccctcgaggt tttcgtcgcg catcggtgta ggtcgaatac ccttgccttt
      721 cagtgcgggg ttttttccta taaagcattt attgtctcga cgcttctcct gcttggttct
      781 ctctcgcact tgcatgacag atttcaagat atcttgcctg tacactcgtc attaaaaaca
      841 gcttattccc ggttattttt ctcaacgaag aaaatggccc ctaagaagaa gagaaaggtg
      901 tcaagaggag tgcaggtgga aaccatctcc ccaggagacg ggcgcacctt ccccaagcgc
      961 ggccagacct gcgtggtgca ctacaccggg atgcttgaag atggaaagaa atttgattcc
     1021 tccagggaca gaaacaagcc ctttaagttt atgctaggca agcaggaggt gatccgaggc
     1081 tgggaagaag gggttgccca gatgagtgtg ggtcagagag ccaaactgac tatatctcca
     1141 gattatgcct atggtgccac tgggcaccca ggcatcatcc caccacatgc cactctcgtc
     1201 ttcgatgtgg agcttctaaa actggaagct agccccagca accccggcgc cagcaacggc
     1261 tccacctctg atgaagtcag gaagaacctg atggacatgt tcagggacag gcaggccttc
     1321 tctgaacaca cctggaagat gctcctgtct gtgtgcagat cctgggctgc ctggtgcaag
     1381 ctgaactgaa ggtcctcacc cgactgggca gctggagtgc agagaacttt gcgcgaccgg
     1441 aagacgctag tgagattggg cacagtggag ctagcgtgcg cgcggtagct ttggcagcac
     1501 tgggattttg tcctctgtac tgcctggctc tcgtgtttat ttttggttag agtgtgcgtt
     1561 gctcggatcc ctgcctggtt ctcgtgctta tttttggtta gatggtgcgt tgttctactt
     1621 tcaatcatcg ttttgttttg tttttcatat ctgttttcca aagaactctg attgtcttca
     1681 tccacccggt tttccccgcc tggtctcgct ttgagattgt ttttattcct ctccggtttt
     1741 tccccattgg gataaaagtg cgtcccgtgt gtctacccct ccagtcccca caagcggctc
     1801 agatgtacag agcacctgct cttctctgac gtgcactttt tgctcttgca gtcttgcgcg
     1861 gcctctagtc tcccctgcca cgcacggatg ctgacgccaa gcagcattct gcgcaggaaa
     1921 gcgtggtatc ccgacgaggc agatcgcggg ctcgcatgta ccggcgacgg acatcctaca
     1981 cacattatcc tcatgtagaa cctcaaagca agggaagaag aaaagggttg acacgttgtt
     2041 atgttttttt ttgtgcctgg gagtaagcgg gcgccgatca agtatcctct caatttcact
     2101 ggcagggcgc gaagtgcgcg ggctcctcgc gtgccgccgc aatgtcagtc gcggctctac
     2161 cagtgctaca gggatgtcgg gtggtgtcgt tgatgatttg gcatcaagtg cacgcgcggc
     2221 cgctttcttc catctgcact cgctctgcgc ttcgttttct tcttcccttg cgcgcgcgtg
     2281 acgccgcgct cctcacctgc gcgccccgcc ccgacttggg tcctctcgtg ctgcaacgga
     2341 cgccgacagc ttccttccac gcatttcgtg cctccgcacc gttctccacc gccagcgcag
     2401 acttgtacat acgcacacac caccatggcc cctaagaaga agagaaaggt gtcaagaatc
     2461 ctctggcatg agatgtggca tgaaggcctg gaagaggcat ctcgtttgta ctttggggaa
     2521 aggaacgtga aaggcatgtt tgaggtgctg gagcccttgc atgctatgat ggaacggggc
     2581 ccccagactc tgaaggaaac atcctttaat caggcctatg gtcgagattt aatggaggcc
     2641 caagagtggt gcaggaagta catgaaatca gggaatgtca aggacctcct ccaagcctgg
     2701 gacctctatt atcatgtgtt ccgacgaatc tcagctagcc ccagcaaccc cggcgccagc
     2761 aacggctcca acaggaaatg gttccctgct gaacctgagg atgtgaggga ctacctcctg
     2821 tacctgcaag ccagaggcct ggctgtgaag accatccaac agcacctggg ccagctcaac
     2881 atgctgcaca ggaggtctgg cctgcctcgc ccttctgact ccaatgctgt gtccctggtg
     2941 atgaggagaa tcagaaagga gaatgtggat gctggggaga gagccaagca ggccctggcc
     3001 tttgaacgca ctgactttga ccaagtcaga tccctgatgg agaactctga cagatgccag
     3061 gacatcagga acctggcctt cctgggcatt gcctacaaca ccctgctgcg cattgccgaa
     3121 attgccagaa tcagagtgaa ggacatctcc cgcaccgatg gtgggagaat gctgatccac
     3181 attggcagga ccaagaccct ggtgtccaca gctggtgtgg agaaggccct gtccctgggg
     3241 gttaccaagc tggtggagag atggatctct gtgtctggtg tggctgatga ccccaacaac
     3301 tacctgttct gccgggtcag aaagaatggt gtggctgccc cttctgccac ctcccaactg
     3361 tccacccgag ccctggaagg gatctttgag gccacccacc gcctgatcta tggtgccaag
     3421 gatgactctg ggcagagata cctggcctgg tctggccact ctgccagagt gggtgctgcc
     3481 agggacatgg ccagggctgg tgtgtccatc cctgaaatca tgcaggctgg tggctggacc
     3541 aatgtgaaca ttgtgatgaa ctacatcaga aacctggact ctgagactgg ggccatggtg
     3601 aggctgctag aggatgggga ctgaagtgtg cgtgagtgtg cgcgtgcgtg ttggtgcgca
     3661 ccgcgctcgc tgtccccgcg gcgcacgcac acctccctcg ctctctcccc ctctttccgt
     3721 gccttctttg tctcccttcc tgtacgctat tttggtttct tgtgcttgct ttatcttgtg
     3781 ggcgggtgtg ggcccgacct gcacgtctgt gacggagtga cgtctagaga gctcgggtcg
     3841 accacacgta cacacgtaca cacacacgtg cgcatgcttg acgcatacgc gacgaattcg
     3901 aaagctcacc tcattcctcc ctcctcacac catcatcggc atccatagag acacgtgcgc
     3961 gtagaacgat acagctcacg cgcacagaga ggcgtgttct ggtcgtgcgc atcatcggca
     4021 cagcactggc ggacgaaact cgcacacagg cacgccgcct cctttcaccc gtcatagata
     4081 gttgaattag acgccctcct cctccctcat catcgccgtc gtcatccggg tccgagcact
     4141 agtatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
     4201 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
     4261 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
     4321 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
     4381 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
     4441 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
     4501 attcgtgaat tgctgccctc tggttatgtg tgggagggct aaagatcttc tagtgatccc
     4561 gcccgcaccc gcgcgcgctc aggccgcgtg tcgtcgtgct ctttccattt tttttttgtg
     4621 tgtggtggtg atttgagctg ctctccgttg tgtgctgggg accctccttc cctcgatctc
     4681 ctcgtgcggg gtctccgacg cgcagacgcg gtgcgtgcga gcgtgcacgt gctgtcgccg
     4741 tggctgtagt tgcgagcgga gagagagaga gagggaggga gggagggcag agggcagagg
     4801 acatgcgggt gggaacgtgc accggcctcg tctcacgcag gcccgggcgg taccctaccc
     4861 agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga
     4921 acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata tcccctatag
     4981 tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct
     5041 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     5101 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     5161 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     5221 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     5281 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     5341 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     5401 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc
     5461 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     5521 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     5581 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     5641 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     5701 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     5761 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     5821 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     5881 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     5941 attggttaat tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag
     6001 ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt
     6061 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6241 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6661 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6721 tgctcacatg tt
//
