LOCUS       sequence-70032-e        7015 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targets TbMCM-BP for Cre-lox mediated knockout in T. brucei.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7015)
  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 7015)
  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: pSY45
FEATURES             Location/Qualifiers
     source          1..7015
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(695..728)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             912..1934
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             912..1934
                     /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"
     regulatory      1971..1980
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1977..3107
                     /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(3114..3147)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(4282..4300)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4283..4300)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4591..4610)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(4821..4840)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4940..4962
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5000..5018)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5086..5190
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5191..5259,5260..6051)
                     /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(5409..5428)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6222..6810
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6711..6730
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6964..6981
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tccccgcgcg ttggccgatt cattaatgca gctgtcgacg ggagaagcgt atggcactga
       61 tgaagctatt tgacaactac cagctgcaaa tagaacaaaa tcagagggag aactgcgaga
      121 gtttagcact gctgaagtca gttgatgatg ccacaatggc gtatgtcttg aagcaaaggg
      181 atcaagaggt ggtagataaa atatgtctat ggaagggtgc atatttagag ggtgataaac
      241 ttttacgggc actactcaag cgctatcaac cgttccagcg gggagacgag aagtttgtct
      301 cccatgtcat tggatacttg aacaaagggc cgcatgtgca agaaccactt gatatatggc
      361 ttgattagtg atgtctgttg ttgttgtatt caggtggcaa tctccacctc aaatgtagtt
      421 agacatcatc agtgccgcct ttatttattt atttttgtat ttttgcttcc ctaccctcta
      481 catttgatac atgcgcactt ttcatgtggg gaacacttcg ctacctgtga ttttatgtac
      541 gttgcaactg tcttcttttt tttttttttt gtggaaatag tagtatactt gcgtgccccc
      601 cttttttttc acagttctac tactggaaag atgagaggat ttggcgaagc tctttttctt
      661 ttttcattta ctttctgaag aagtgcagaa gcttataact tcgtatagca tacattatac
      721 gaagttatac cggtacacgt tctcgtcggc cggccgctgc acgcgccttc gagttttttt
      781 tccttttccc catttttttc aacttgaaga cttcaattac accaaaaaat aaaattcaca
      841 aacttggtat tcctttgtgt tacattcttg atcgctcgca ctgacattac agatcttact
      901 gttggtaaat gatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
      961 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     1021 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     1081 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     1141 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     1201 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     1261 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     1321 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     1381 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     1441 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     1501 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     1561 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     1621 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     1681 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     1741 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     1801 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     1861 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     1921 cgagggcaaa ggaatctaga gaagtccata ctaaccagga cccacttgac gccaccatgg
     1981 cctcgtaccc cggccatcaa cacgcgtctg cgttcgacca ggctgcgcgt tctcgcggcc
     2041 atagcaaccg acgtacggcg ttgcgccctc gccggcagca agaagccacg gaagtccgcc
     2101 cggagcagaa aatgcccacg ctactgcggg tttatataga cggtccccac gggatgggga
     2161 aaaccaccac cacgcaactg ctggtggccc tgggttcgcg cgacgatatc gtctacgtac
     2221 ccgagccgat gacttactgg cgggtgctgg gggcttccga gacaatcgcg aacatctaca
     2281 ccacacaaca ccgcctcgac cagggtgaga tatcggccgg ggacgcggcg gtggtaatga
     2341 caagcgccca gataacaatg ggcatgcctt atgccgtgac cgacgccgtt ctggctcctc
     2401 atatcggggg ggaggctggg agctcacatg ccccgccccc ggccctcacc ctcatcttcg
     2461 accgccatcc catcgccgcc ctcctgtgct acccggccgc gcggtacctt atgggcagca
     2521 tgacccccca ggccgtgctg gcgttcgtgg ccctcatccc gccgaccttg cccggcacca
     2581 acatcgtgct tggggccctt ccggaggaca gacacatcga ccgcctggcc aaacgccagc
     2641 gccccggcga gcggctggac ctggctatgc tggctgcgat tcgccgcgtt tacgggctac
     2701 ttgccaatac ggtgcggtat ctgcagtgcg gcgggtcgtg gcgggaggac tggggacagc
     2761 tttcggggac ggccgtgccg ccccagggtg ccgagcccca gagcaacgcg ggcccacgac
     2821 cccatatcgg ggacacgtta tttaccctgt ttcgggcccc cgagttgctg gcccccaacg
     2881 gcgacctgta taacgtgttt gcctgggcct tggacgtctt ggccaaacgc ctccgttcca
     2941 tgcacgtctt tatcctggat tacgaccaat cgcccgccgg ctgccgggac gccctgctgc
     3001 aacttacctc cgggatggtc cagacccacg tcaccacccc cggctccata ccgacgatat
     3061 gcgacctggc gcgcacgttt gcccgggaga tgggggaggc taactgagaa ttcataactt
     3121 cgtatagcat acattatacg aagttatgga tcctggctgc ggtgctgggt ggatcacaaa
     3181 gcaagtgtgt gcagcatacc caacatcctt cttcccaccc gttggttctt cgtttaccta
     3241 ctctgttata gtagtaatag atgccgctgt cttggttaag cacttaacct gtgactctcc
     3301 ctgctggtta gtgctgtact tcctacacaa acatacccgc ctctaacttg aatcgttctc
     3361 tttcaactcg ttttgcattc ttttctcttt ctctcactct ctctcacatt catttgctgc
     3421 atgcgcatat tatcctgact actgatgcat acaatttata ttgaagaagg taaatagagc
     3481 atatcaacag tttaaaaaaa aggccaaata acgtaaagta acatattaca tttttttttt
     3541 ttgaaagtgt cgttggatct cgtgacaaca gaataacaca gaaggaaaaa gcacttaaca
     3601 ttttggacat gtcacttgtg gaggttttgc cgaactactt cactctttcg aaggactcac
     3661 cccttcggaa gaagttcgaa aaggtgtaca aatggtattc accagccttt tcgcctcacg
     3721 atgttccgcg ctttgctgag gtggggaata tcaccgaaaa tcctgaagtg atgcgcggca
     3781 ttcgagactt cttcgttgac cgctataaaa atcttcagca gcccatcacc cacattttgg
     3841 ggtttgactc tcgtggtttt ctgcttggac caatgattgc agtggagctg aacgtgcctt
     3901 ttgtactgat aaggaaggcg aacaaaatcg ctggtgttat cattaagagt gaaccttaca
     3961 cgaaggaata cgccgcagaa tcagaggagt gcatgaccgt tcgcttcggg agctttgaca
     4021 agaattctcg cgttgttctc atcgatgatg tgattgccac tggtggtacc atgcttgcgg
     4081 gagtgcaact tgttgatgca tgtggtgcca ctcttgtgga agtggcgggt attctcggcc
     4141 tcactttcct caaaggaacg caaactagta aaaatcacta gtgaattcgc ggccgccagg
     4201 ttggttagga aggggggatg atgtaaaaga agaaaatggg gggattcgag cccccttgca
     4261 ggcatgcaag ctagcttgta ttctatagtg tcacctaaat cgtatgtgta tgatacataa
     4321 ggttatgtat taattgtagc cgcgttctaa cgacaatatg tacaagccta attgtgtagc
     4381 atctggctta ctgaagcaga ccctatcatc tctctcgtaa actgccgtca gagtcggttt
     4441 ggttggacga accttctgag tttctggtaa cgccgttccg caccccggaa atggtcagcg
     4501 aaccaatcag cagggtcatc gctagccaga tcctctacgc cggacgcatc gtggccggca
     4561 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     4621 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
     4681 ccgtggccgg gggactgttg ggcgccatct ccttgcacca ttccttgcgg cggcggtgct
     4741 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     4801 tcgatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     4861 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4921 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4981 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5041 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5101 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5161 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5221 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5281 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5341 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5401 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5461 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5521 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5581 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5641 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5701 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5761 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5821 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5881 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5941 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6001 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6061 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6121 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6181 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6241 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6301 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6361 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6421 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6481 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6541 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6601 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6661 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6721 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6781 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6841 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6901 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6961 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctc
//
