LOCUS       sequence-71676-e        4603 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3XMYC-intergenic region of TUB-HYG (modified from PMID 16269191).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4603)
  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 4603)
  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: pDS67
FEATURES             Location/Qualifiers
     source          1..4603
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             113..142
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             155..184
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             197..226
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             610..1632
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             610..1632
                     /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"
     promoter        complement(1725..1743)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1726..1743)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1761..1777)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1761..1777)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1774..1796)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1785..1801
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1809..1815,1816..1833,1834..1839))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1854..1875
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1992..2009)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2163..2751)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2243..2262)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2922..3713,3714..3782))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3545..3564
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3783..3887)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3968..4423)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4105..4126)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4317..4336
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4549..4571
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4563..4580
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4564..4580
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(4587..4603,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4587..4603,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 ttttagatct ggtaccgggc cccccctcga gggcggccga tcgagatccg aggagcagaa
      121 gctgatctcg gaggaggatc tgctgagatc cgaggagcag aagctgatct cggaggagga
      181 tctgctgaga tccgaggagc agaagctgat ctcagaggag gatctgctgt gatctagtgc
      241 ggccgtcgac aaagtgtgac aacgtcgcac catgtgtagg ttttcattta tgttctttct
      301 ttctttcttt tgtgaatttg ttttctgtct caaatgtttt taattcgctt gggacctatg
      361 tttttcttgt ttttttgctc accctttgtg taggaggcac cctgtcacgt ctgtggttgc
      421 gtgtatgcct tccttcccct tattcgcttc ttcctgtcgt gtcacacctc tttctccctc
      481 tccctttccg ccttttcttt caatcttgtt ttctcgacca gccctactag aggagaaaga
      541 atagtaaccc tttcatcaaa gaaaatagtt caaacgaatt gctagcttaa gcttgatcta
      601 gaactagtga tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
      661 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
      721 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
      781 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
      841 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
      901 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
      961 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     1021 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     1081 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     1141 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     1201 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     1261 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     1321 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     1381 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     1441 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     1501 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     1561 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     1621 agggcaaagg aatcttctag aaaaaatcac tagtgaattc gcggccgcct gcaggtcgac
     1681 catatgggag agctcccaac gcgttggatg catagcttga gtattctata gtgtcaccta
     1741 aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1801 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1861 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1921 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1981 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2041 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2101 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2161 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2221 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2281 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2341 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2401 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2461 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2521 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2581 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     2641 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2701 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2761 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2821 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2881 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2941 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3001 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3061 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3121 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3181 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgg cattgctaca
     3241 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3301 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3361 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3421 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3481 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3541 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3601 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3661 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3721 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3781 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3841 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3901 aaagtgccac ctgtatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     3961 tcaggcgaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaatat ttgttaaatc
     4021 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     4081 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     4141 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     4201 tcacccaaat caagtttttt gcggtcgagg tgccgtaaag ctctaaatcg gaaccctaaa
     4261 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     4321 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     4381 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc
     4441 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     4501 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     4561 gttgtaaaac gacggccagt gaattgtaat acgactcact ata
//
