LOCUS       sequence-71698-e        5178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP-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 5178)
  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 5178)
  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: pDS68
FEATURES             Location/Qualifiers
     source          1..5178
                     /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             join(2990..2992,2993..2995,2996..3706)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2990..2992,2993..2995,2996..3706)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3035..3056)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3296..3315)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3643..3664
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4083..5105
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4083..5105
                     /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      5133..5142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      5137..5146
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(join(5176..5178,1..17))
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(join(5177..5178,1..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 cccctcgaga tggtgagcaa
     3001 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3061 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     3121 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     3181 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     3241 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     3301 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     3361 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3421 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     3481 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3541 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     3601 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3661 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagcgtt accgctagtc
     3721 gacaaagtgt gacaacgtcg caccatgtgt aggttttcat ttatgttctt tctttctttc
     3781 ttttgtgaat ttgttttctg tctcaaatgt ttttaattcg cttgggacct atgtttttct
     3841 tgtttttttg ctcacccttt gtgtaggagg caccctgtca cgtctgtggt tgcgtgtatg
     3901 ccttccttcc ccttattcgc ttcttcctgt cgtgtcacac ctctttctcc ctctcccttt
     3961 ccgccttttc tttcaatctt gttttctcga ccagccctac tagaggagaa agaatagtaa
     4021 ccctttcatc aaagaaaata gttcaaacga attgctagct taagcttgat ctagaactag
     4081 tgatgaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     4141 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     4201 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     4261 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     4321 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     4381 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     4441 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     4501 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     4561 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     4621 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     4681 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     4741 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     4801 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     4861 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     4921 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     4981 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     5041 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     5101 aggaatcttc tagaaaaaat cgaattcccg cggccgccat ggcggccggg agcatgcgac
     5161 gtcgggccca attcgccc
//
