LOCUS       sequence-70046-e        5820 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Targets beta-Tubulin for Cre-lox mediated knockout in T. brucei, 
            Puromycin//Gancyclovir selectable.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5820)
  AUTHORS   Kim HS, Li Z, Boothroyd C, Cross GA
  TITLE     Strategies to construct null and conditional null Trypanosoma brucei
            mutants using Cre-recombinase and loxP.
  JOURNAL   Mol Biochem Parasitol. 2013 Aug 13;191(1):16-19. doi: 
            10.1016/j.molbiopara.2013.08.001.
  PUBMED    23954366
REFERENCE   2  (bases 1 to 5820)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molbiopara.2013.08"; journalName: "Mol Biochem 
            Parasitol"; date: "2013-08-13- 13"; volume: "191"; issue: "1"; 
            pages: "16-19"
COMMENT     Sequence Label: pHJ18
FEATURES             Location/Qualifiers
     source          1..5820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1109..1142)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1311..1907
                     /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(1311..1330)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1807..1827
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     regulatory      1944..1953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1950..3080
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     protein_bind    complement(3087..3120)
                     /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(3365..3384)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3595..3614)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3714..3736
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3774..3792)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3860..3964
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3965..4033,4034..4825)
                     /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     complement(4183..4202)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4996..5584
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5485..5504
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5738..5755
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctgaccgtat catgatgact ttctccatca tcccatcccc caaggtgtcc gacactgtcg
       61 tcgagccgta caatacgact ctctccgtgc accaacttgt ggaaaactcc gatgagtcga
      121 tgtgcattga caacgaggca ctgtacgata tttgcttccg caccctgaaa ctgacaacac
      181 caacgttcgg tgacctgaac cacttggtgt ctgctgttgt gtccggcgtc acctgctgcc
      241 tgcgcttccc tggtcagttg aactctgacc tccgtaagtt ggctgtgaac cttgtcccat
      301 tcccgcgtct gcacttcttc atgatgggct tcgccccgct gaccagccgc ggctcgcagc
      361 agtaccgcgg tctctccgtg cccgagctaa cgcagcagat gttcgatgcg aaaaacatga
      421 tgcaagctgc agatcctcgt cacggccgct acctgacagc gtctgcactc ttccgcggcc
      481 gcatgtcgac gaaggaggtt gatgagcaga tgctgaacgt gcagaacaag aactcgtcct
      541 acttcattga gtggatcgat cccgaacaac atcaagtcct ctgtttgcga tatcccaccc
      601 aagggactca agatggctgt caccttcatt ggcaacaaca cctgcatcca ggagatgttc
      661 cgccgtgtgg gagagcagtt caccctcatg ttccgtcgca aggcgttctt gcactggtac
      721 actggcgagg gtatggacga gatggaattc acggaggcag agtccaacat gaacgatctc
      781 gtgtctgagt accagcagta ccaggatgcc acgattgagg aggagggcga gttcgacgag
      841 gaggagcaat actagacgcg gacggggcat ttcccgttcg tcattagcag taggtaatga
      901 agatgctcga gggtgctcaa gctgtgtagc gcacgcgttt ccttacatat ttctctaaca
      961 ggcacggaag cctaacaaat acacttggct tatttttttg ccccctcatg tcttgtacaa
     1021 atatttgcga tagcttagct atcagccaca ttaatcaaac aagtatacca acaagcccga
     1081 aaacataaac tcaactgcaa cgaagcttat aacttcgtat agcatacatt atacgaagtt
     1141 ataccggtac acgttctcgt cggccggccg ctgcacgcgc cttcgagttt tttttccttt
     1201 tccccatttt tttcaacttg aagacttcaa ttacaccaaa aaataaaatt cacaaacttg
     1261 gtattccttt gtgttacatt cttgatcgct cgcactgaca ttacagatct atgaccgagt
     1321 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     1381 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccagac cgccacatcg
     1441 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     1501 tgtgggtcgc ggacgacggc gcagcagtgg cggtctggac cacgccggag agcgtcgaag
     1561 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     1621 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     1681 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     1741 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     1801 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     1861 tgcccgaagg accgcgcact tggtgcatga cccgcaagcc cggtgcctct agagaagtcc
     1921 atactaacca ggacccactt gacgccacca tggcctcgta ccccggccat caacacgcgt
     1981 ctgcgttcga ccaggctgcg cgttctcgcg gccatagcaa ccgacgtacg gcgttgcgcc
     2041 ctcgccggca gcaagaagcc acggaagtcc gcccggagca gaaaatgccc acgctactgc
     2101 gggtttatat agacggtccc cacgggatgg ggaaaaccac caccacgcaa ctgctggtgg
     2161 ccctgggttc gcgcgacgat atcgtctacg tacccgagcc gatgacttac tggcgggtgc
     2221 tgggggcttc cgagacaatc gcgaacatct acaccacaca acaccgcctc gaccagggtg
     2281 agatatcggc cggggacgcg gcggtggtaa tgacaagcgc ccagataaca atgggcatgc
     2341 cttatgccgt gaccgacgcc gttctggctc ctcatatcgg gggggaggct gggagctcac
     2401 atgccccgcc cccggccctc accctcatct tcgaccgcca tcccatcgcc gccctcctgt
     2461 gctacccggc cgcgcggtac cttatgggca gcatgacccc ccaggccgtg ctggcgttcg
     2521 tggccctcat cccgccgacc ttgcccggca ccaacatcgt gcttggggcc cttccggagg
     2581 acagacacat cgaccgcctg gccaaacgcc agcgccccgg cgagcggctg gacctggcta
     2641 tgctggctgc gattcgccgc gtttacgggc tacttgccaa tacggtgcgg tatctgcagt
     2701 gcggcgggtc gtggcgggag gactggggac agctttcggg gacggccgtg ccgccccagg
     2761 gtgccgagcc ccagagcaac gcgggcccac gaccccatat cggggacacg ttatttaccc
     2821 tgtttcgggc ccccgagttg ctggccccca acggcgacct gtataacgtg tttgcctggg
     2881 ccttggacgt cttggccaaa cgcctccgtt ccatgcacgt ctttatcctg gattacgacc
     2941 aatcgcccgc cggctgccgg gacgccctgc tgcaacttac ctccgggatg gtccagaccc
     3001 acgtcaccac ccccggctcc ataccgacga tatgcgacct ggcgcgcacg tttgcccggg
     3061 agatggggga ggctaactga gaattcataa cttcgtatag catacattat acgaagttat
     3121 ggatcctgcc catttagtta gttggctttt cccttgtctc gtgtcttttc cgtggaaagg
     3181 ttcccggagt aatctgatgg cacagcaggg aggtgcgcct gcaggttggt taggaagggg
     3241 ggatgatgta aaagaagaaa atggggggat tcgagccccc ttgcaggcat gcaagctagc
     3301 cagatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
     3361 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
     3421 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc
     3481 atctccttgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     3541 gcttcctaat gcaggagtcg cataagggag agcgtcgata tggtgcactc tcagtacaat
     3601 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     3661 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3721 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     3781 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     3841 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3901 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3961 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4021 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4081 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4141 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4201 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4261 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4321 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4381 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4441 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4501 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4561 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4621 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4681 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4741 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4801 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4861 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4921 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4981 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5041 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5101 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     5161 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5221 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5281 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5341 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5401 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5461 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5521 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5581 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5641 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     5701 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     5761 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
//
