LOCUS       sequence-70049-e        5599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targets beta-Tubulin for Cre-lox mediated knockout in T. brucei, 
            Bleomycin/Gancyclovir selectable.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5599)
  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 5599)
  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"; 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: pHJ42
FEATURES             Location/Qualifiers
     source          1..5599
                     /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..1686
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     regulatory      1723..1732
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1729..2859
                     /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(2866..2899)
                     /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(3144..3163)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3374..3393)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3493..3515
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3553..3571)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3639..3743
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3744..3812,3813..4604)
                     /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(3962..3981)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4775..5363
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5264..5283
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5517..5534
                     /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 caccatggcc
     1321 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc
     1381 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc
     1441 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc
     1501 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg
     1561 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg
     1621 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag
     1681 caggactcta gagaagtcca tactaaccag gacccacttg acgccaccat ggcctcgtac
     1741 cccggccatc aacacgcgtc tgcgttcgac caggctgcgc gttctcgcgg ccatagcaac
     1801 cgacgtacgg cgttgcgccc tcgccggcag caagaagcca cggaagtccg cccggagcag
     1861 aaaatgccca cgctactgcg ggtttatata gacggtcccc acgggatggg gaaaaccacc
     1921 accacgcaac tgctggtggc cctgggttcg cgcgacgata tcgtctacgt acccgagccg
     1981 atgacttact ggcgggtgct gggggcttcc gagacaatcg cgaacatcta caccacacaa
     2041 caccgcctcg accagggtga gatatcggcc ggggacgcgg cggtggtaat gacaagcgcc
     2101 cagataacaa tgggcatgcc ttatgccgtg accgacgccg ttctggctcc tcatatcggg
     2161 ggggaggctg ggagctcaca tgccccgccc ccggccctca ccctcatctt cgaccgccat
     2221 cccatcgccg ccctcctgtg ctacccggcc gcgcggtacc ttatgggcag catgaccccc
     2281 caggccgtgc tggcgttcgt ggccctcatc ccgccgacct tgcccggcac caacatcgtg
     2341 cttggggccc ttccggagga cagacacatc gaccgcctgg ccaaacgcca gcgccccggc
     2401 gagcggctgg acctggctat gctggctgcg attcgccgcg tttacgggct acttgccaat
     2461 acggtgcggt atctgcagtg cggcgggtcg tggcgggagg actggggaca gctttcgggg
     2521 acggccgtgc cgccccaggg tgccgagccc cagagcaacg cgggcccacg accccatatc
     2581 ggggacacgt tatttaccct gtttcgggcc cccgagttgc tggcccccaa cggcgacctg
     2641 tataacgtgt ttgcctgggc cttggacgtc ttggccaaac gcctccgttc catgcacgtc
     2701 tttatcctgg attacgacca atcgcccgcc ggctgccggg acgccctgct gcaacttacc
     2761 tccgggatgg tccagaccca cgtcaccacc cccggctcca taccgacgat atgcgacctg
     2821 gcgcgcacgt ttgcccggga gatgggggag gctaactgag aattcataac ttcgtatagc
     2881 atacattata cgaagttatg gatcctgccc atttagttag ttggcttttc ccttgtctcg
     2941 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg
     3001 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccct
     3061 tgcaggcatg caagctagcc agatcctcta cgccggacgc atcgtggccg gcatcaccgg
     3121 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc
     3181 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
     3241 cgggggactg ttgggcgcca tctccttgca ccattccttg cggcggcggt gctcaacggc
     3301 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgatat
     3361 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     3421 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3481 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3541 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3601 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3661 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3721 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3781 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3841 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3901 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3961 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     4021 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4081 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4141 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4201 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4261 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4321 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4381 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4441 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4501 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4561 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4621 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4681 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4741 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4801 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4861 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4921 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4981 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5041 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5101 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5161 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5221 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5281 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5341 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5401 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5461 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5521 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5581 ggccgattca ttaatgcag
//
