LOCUS       sequence-70047-e        6092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targets beta-Tubulin for Cre-lox mediated knockout in T. brucei, 
            Neomycin/Gancyclovir selectable.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6092)
  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 6092)
  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: pHJ22
FEATURES             Location/Qualifiers
     source          1..6092
                     /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)."
     primer_bind     complement(1442..1461)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2052..2071
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2216..2225
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2222..3352
                     /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(3359..3392)
                     /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(3637..3656)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3867..3886)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3986..4008
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4046..4064)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4132..4236
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4237..4305,4306..5097)
                     /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(4455..4474)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5268..5856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5757..5776
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6010..6027
                     /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 cgaatatgcg
     1321 cgaaatcgtc tgcgttcagg ctggccaatg cggtaaccag atcggctcaa agttctggga
     1381 ggtgatccgg ccaagcttgg atggattgca cgcaggttct ccggccgctt gggtggagag
     1441 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     1501 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     1561 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     1621 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     1681 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     1741 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     1801 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     1861 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     1921 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     1981 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     2041 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     2101 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     2161 ccttcttgac gagttcttct ctagagaagt ccatactaac caggacccac ttgacgccac
     2221 catggcctcg taccccggcc atcaacacgc gtctgcgttc gaccaggctg cgcgttctcg
     2281 cggccatagc aaccgacgta cggcgttgcg ccctcgccgg cagcaagaag ccacggaagt
     2341 ccgcccggag cagaaaatgc ccacgctact gcgggtttat atagacggtc cccacgggat
     2401 ggggaaaacc accaccacgc aactgctggt ggccctgggt tcgcgcgacg atatcgtcta
     2461 cgtacccgag ccgatgactt actggcgggt gctgggggct tccgagacaa tcgcgaacat
     2521 ctacaccaca caacaccgcc tcgaccaggg tgagatatcg gccggggacg cggcggtggt
     2581 aatgacaagc gcccagataa caatgggcat gccttatgcc gtgaccgacg ccgttctggc
     2641 tcctcatatc gggggggagg ctgggagctc acatgccccg cccccggccc tcaccctcat
     2701 cttcgaccgc catcccatcg ccgccctcct gtgctacccg gccgcgcggt accttatggg
     2761 cagcatgacc ccccaggccg tgctggcgtt cgtggccctc atcccgccga ccttgcccgg
     2821 caccaacatc gtgcttgggg cccttccgga ggacagacac atcgaccgcc tggccaaacg
     2881 ccagcgcccc ggcgagcggc tggacctggc tatgctggct gcgattcgcc gcgtttacgg
     2941 gctacttgcc aatacggtgc ggtatctgca gtgcggcggg tcgtggcggg aggactgggg
     3001 acagctttcg gggacggccg tgccgcccca gggtgccgag ccccagagca acgcgggccc
     3061 acgaccccat atcggggaca cgttatttac cctgtttcgg gcccccgagt tgctggcccc
     3121 caacggcgac ctgtataacg tgtttgcctg ggccttggac gtcttggcca aacgcctccg
     3181 ttccatgcac gtctttatcc tggattacga ccaatcgccc gccggctgcc gggacgccct
     3241 gctgcaactt acctccggga tggtccagac ccacgtcacc acccccggct ccataccgac
     3301 gatatgcgac ctggcgcgca cgtttgcccg ggagatgggg gaggctaact gagaattcat
     3361 aacttcgtat agcatacatt atacgaagtt atggatcctg cccatttagt tagttggctt
     3421 ttcccttgtc tcgtgtcttt tccgtggaaa ggttcccgga gtaatctgat ggcacagcag
     3481 ggaggtgcgc ctgcaggttg gttaggaagg ggggatgatg taaaagaaga aaatgggggg
     3541 attcgagccc ccttgcaggc atgcaagcta gccagatcct ctacgccgga cgcatcgtgg
     3601 ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     3661 gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg
     3721 caggccccgt ggccggggga ctgttgggcg ccatctcctt gcaccattcc ttgcggcggc
     3781 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     3841 agagcgtcga tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3901 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3961 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4021 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4081 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4141 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4201 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4261 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4321 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4381 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4441 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     4501 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4561 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4621 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4681 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4741 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4801 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4861 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4921 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4981 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5041 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5101 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5161 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5221 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5281 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5341 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5401 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5461 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5521 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5581 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5641 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5701 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5761 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5821 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5881 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5941 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6001 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     6061 ctccccgcgc gttggccgat tcattaatgc ag
//
