LOCUS       sequence-70059-e        4533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3XHA-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 4533)
  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 4533)
  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: pDS66
FEATURES             Location/Qualifiers
     source          1..4533
                     /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             92..172
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             540..1562
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             540..1562
                     /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(1655..1673)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1656..1673)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1691..1707)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1691..1707)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1704..1726)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1715..1731
                     /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(1739..1745,1746..1763,1764..1769))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1784..1805
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1922..1939)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2093..2681)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2173..2192)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2852..3643,3644..3712))
                     /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     3475..3494
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3713..3817)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3898..4353)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4035..4056)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4247..4266
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4479..4501
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4493..4510
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4494..4510
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(4517..4533,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4517..4533,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 ttttagatct ggtaccgggc cccccctcga gtacccttac gatgtgcctg attacgcgta
      121 cccatacgac gtgccagact acgcataccc gtacgatgtg cccgattacg catagtcgac
      181 aaagtgtgac aacgtcgcac catgtgtagg ttttcattta tgttctttct ttctttcttt
      241 tgtgaatttg ttttctgtct caaatgtttt taattcgctt gggacctatg tttttcttgt
      301 ttttttgctc accctttgtg taggaggcac cctgtcacgt ctgtggttgc gtgtatgcct
      361 tccttcccct tattcgcttc ttcctgtcgt gtcacacctc tttctccctc tccctttccg
      421 ccttttcttt caatcttgtt ttctcgacca gccctactag aggagaaaga atagtaaccc
      481 tttcatcaaa gaaaatagtt caaacgaatt gctagcttaa gcttgatcta gaactagtga
      541 tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
      601 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
      661 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
      721 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
      781 gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
      841 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
      901 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
      961 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     1021 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     1081 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     1141 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     1201 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     1261 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     1321 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     1381 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     1441 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     1501 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     1561 aatcttctag aaaaaatcac tagtgaattc gcggccgcct gcaggtcgac catatgggag
     1621 agctcccaac gcgttggatg catagcttga gtattctata gtgtcaccta aatagcttgg
     1681 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1741 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1801 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1861 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1921 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1981 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2041 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2101 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2161 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2221 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2281 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2341 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2401 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2461 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2521 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2581 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2641 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2701 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2761 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2821 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     2881 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     2941 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3001 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3061 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3121 taagtagttc gccagttaat agtttgcgca acgttgttgg cattgctaca ggcatcgtgg
     3181 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3241 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3301 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3361 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3421 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3481 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3541 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3601 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3661 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3721 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3781 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     3841 ctgtatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgaaa
     3901 ttgtaaacgt taatattttg ttaaaattcg cgttaaatat ttgttaaatc agctcatttt
     3961 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     4021 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     4081 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcacccaaat
     4141 caagtttttt gcggtcgagg tgccgtaaag ctctaaatcg gaaccctaaa gggagccccc
     4201 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     4261 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     4321 ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg
     4381 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     4441 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     4501 gacggccagt gaattgtaat acgactcact ata
//
