LOCUS       sequence-70052-e        5118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targets beta-Tubulin for Cre-lox mediated knockout in T. brucei, 
            Hygromycin selectable.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5118)
  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 5118)
  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: pHJ32
FEATURES             Location/Qualifiers
     source          1..5118
                     /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             1326..2348
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1326..2348
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     protein_bind    complement(2385..2418)
                     /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(2663..2682)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(2893..2912)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3012..3034
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3072..3090)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3158..3262
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3263..3331,3332..4123)
                     /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(3481..3500)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4294..4882
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4783..4802
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5036..5053
                     /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 tactgttggt
     1321 aaatgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     1381 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     1441 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     1501 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     1561 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     1621 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     1681 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     1741 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     1801 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     1861 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     1921 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     1981 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     2041 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     2101 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     2161 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     2221 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     2281 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     2341 caaaggaatc tagccgggag atgggggagg ctaactgaga attcataact tcgtatagca
     2401 tacattatac gaagttatgg atcctgccca tttagttagt tggcttttcc cttgtctcgt
     2461 gtcttttccg tggaaaggtt cccggagtaa tctgatggca cagcagggag gtgcgcctgc
     2521 aggttggtta ggaagggggg atgatgtaaa agaagaaaat ggggggattc gagccccctt
     2581 gcaggcatgc aagctagcca gatcctctac gccggacgca tcgtggccgg catcaccggc
     2641 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     2701 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     2761 gggggactgt tgggcgccat ctccttgcac cattccttgc ggcggcggtg ctcaacggcc
     2821 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgatatg
     2881 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     2941 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3001 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3061 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3121 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3181 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3241 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3301 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3361 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3421 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3481 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3541 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3601 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3661 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3721 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3781 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3841 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3901 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3961 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4021 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4081 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4141 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4201 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4261 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4321 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4381 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4441 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4501 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4561 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4621 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4681 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4741 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4801 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4861 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4921 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4981 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5041 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     5101 gccgattcat taatgcag
//
