LOCUS       sequence-70051-e        5623 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Targets beta-Tubulin for Cre-lox mediated knockout in T. brucei, 
            Blasticidin/Gancyclovir selectable.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5623)
  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 5623)
  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: pHJ41
FEATURES             Location/Qualifiers
     source          1..5623
                     /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             1315..1710
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      1747..1756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1753..2883
                     /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(2890..2923)
                     /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(3168..3187)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3398..3417)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3517..3539
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3577..3595)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3663..3767
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3768..3836,3837..4628)
                     /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(3986..4005)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4799..5387
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5288..5307
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5541..5558
                     /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 cagcatggcc
     1321 aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc
     1381 atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc
     1441 ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg
     1501 ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag
     1561 aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct
     1621 gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa
     1681 ttgctgccct ctggttatgt gtgggagggc tctagagaag tccatactaa ccaggaccca
     1741 cttgacgcca ccatggcctc gtaccccggc catcaacacg cgtctgcgtt cgaccaggct
     1801 gcgcgttctc gcggccatag caaccgacgt acggcgttgc gccctcgccg gcagcaagaa
     1861 gccacggaag tccgcccgga gcagaaaatg cccacgctac tgcgggttta tatagacggt
     1921 ccccacggga tggggaaaac caccaccacg caactgctgg tggccctggg ttcgcgcgac
     1981 gatatcgtct acgtacccga gccgatgact tactggcggg tgctgggggc ttccgagaca
     2041 atcgcgaaca tctacaccac acaacaccgc ctcgaccagg gtgagatatc ggccggggac
     2101 gcggcggtgg taatgacaag cgcccagata acaatgggca tgccttatgc cgtgaccgac
     2161 gccgttctgg ctcctcatat cgggggggag gctgggagct cacatgcccc gcccccggcc
     2221 ctcaccctca tcttcgaccg ccatcccatc gccgccctcc tgtgctaccc ggccgcgcgg
     2281 taccttatgg gcagcatgac cccccaggcc gtgctggcgt tcgtggccct catcccgccg
     2341 accttgcccg gcaccaacat cgtgcttggg gcccttccgg aggacagaca catcgaccgc
     2401 ctggccaaac gccagcgccc cggcgagcgg ctggacctgg ctatgctggc tgcgattcgc
     2461 cgcgtttacg ggctacttgc caatacggtg cggtatctgc agtgcggcgg gtcgtggcgg
     2521 gaggactggg gacagctttc ggggacggcc gtgccgcccc agggtgccga gccccagagc
     2581 aacgcgggcc cacgacccca tatcggggac acgttattta ccctgtttcg ggcccccgag
     2641 ttgctggccc ccaacggcga cctgtataac gtgtttgcct gggccttgga cgtcttggcc
     2701 aaacgcctcc gttccatgca cgtctttatc ctggattacg accaatcgcc cgccggctgc
     2761 cgggacgccc tgctgcaact tacctccggg atggtccaga cccacgtcac cacccccggc
     2821 tccataccga cgatatgcga cctggcgcgc acgtttgccc gggagatggg ggaggctaac
     2881 tgagaattca taacttcgta tagcatacat tatacgaagt tatggatcct gcccatttag
     2941 ttagttggct tttcccttgt ctcgtgtctt ttccgtggaa aggttcccgg agtaatctga
     3001 tggcacagca gggaggtgcg cctgcaggtt ggttaggaag gggggatgat gtaaaagaag
     3061 aaaatggggg gattcgagcc cccttgcagg catgcaagct agccagatcc tctacgccgg
     3121 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     3181 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     3241 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcaccattc
     3301 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
     3361 tcgcataagg gagagcgtcg atatggtgca ctctcagtac aatctgctct gatgccgcat
     3421 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3481 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3541 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     3601 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     3661 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3721 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3781 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3841 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     3901 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3961 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4021 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4081 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4141 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4201 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4261 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4321 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4381 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4441 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     4501 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     4561 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4621 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4681 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4741 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4801 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4861 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4921 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4981 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5041 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5101 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5161 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5221 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5281 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5341 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5401 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5461 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     5521 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     5581 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cag
//
