LOCUS       sequence-71699-e        4515 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLAG-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 4515)
  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 4515)
  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: pDS69
FEATURES             Location/Qualifiers
     source          1..4515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..41)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(24..40)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(33..55)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      181..636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     478..499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        717..821
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(822..890,891..1682)
                     /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     complement(1040..1059)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1853..2441
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2342..2361
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2595..2612
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2729..2750
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2765..2770,2771..2788,2789..2795)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2803..2819
                     /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)."
     primer_bind     2808..2830
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2827..2843
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2827..2843
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2861..2879
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2861..2878
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             3029..3052
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3420..4442
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3420..4442
                     /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      4470..4479
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4474..4483
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(join(4513..4515,1..17))
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(join(4514..4515,1..17))
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
       61 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
      121 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
      181 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      241 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      301 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      361 gagctttacg gcacctcgac cgcaaaaaac ttgatttggg tgatggttca cgtagtgggc
      421 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      481 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      541 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaatattta
      601 acgcgaattt taacaaaata ttaacgttta caatttcgcc tgatgcggta ttttctcctt
      661 acgcatctgt gcggtatttc acaccgcata caggtggcac ttttcgggga aatgtgcgcg
      721 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
      781 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
      841 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
      901 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
      961 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1021 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1081 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1141 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1201 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1261 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1321 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atgccaacaa
     1381 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     1441 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     1501 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     1561 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     1621 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     1681 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     1741 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     1801 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     1861 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     1921 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     1981 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2041 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2101 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2161 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2221 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2281 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2341 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2401 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     2461 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     2521 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     2581 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     2641 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     2701 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     2761 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     2821 ttcacacagg aaacagctat gaccatgatt acgccaagct atttaggtga cactatagaa
     2881 tactcaagct atgcatccaa cgcgttggga gctctcccat atggtcgacc tgcaggcggc
     2941 cgcgaattca ctagtgattt ttagatctgg taccgggccc cccctcgagt acaaggatca
     3001 cgatggtgat tacaaggatc acgatattga ttacaaggac gatgacgata agtagtcgac
     3061 aaagtgtgac aacgtcgcac catgtgtagg ttttcattta tgttctttct ttctttcttt
     3121 tgtgaatttg ttttctgtct caaatgtttt taattcgctt gggacctatg tttttcttgt
     3181 ttttttgctc accctttgtg taggaggcac cctgtcacgt ctgtggttgc gtgtatgcct
     3241 tccttcccct tattcgcttc ttcctgtcgt gtcacacctc tttctccctc tccctttccg
     3301 ccttttcttt caatcttgtt ttctcgacca gccctactag aggagaaaga atagtaaccc
     3361 tttcatcaaa gaaaatagtt caaacgaatt gctagcttaa gcttgatcta gaactagtga
     3421 tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
     3481 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
     3541 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
     3601 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     3661 gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     3721 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     3781 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     3841 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     3901 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     3961 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     4021 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     4081 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     4141 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     4201 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     4261 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     4321 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     4381 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     4441 aatcttctag aaaaaatcga attcccgcgg ccgccatggc ggccgggagc atgcgacgtc
     4501 gggcccaatt cgccc
//
