LOCUS       sequence-75241-e        6094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express CLIP and FLAG-tagged human CD59, a GPI-AP in mammmalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6094)
  AUTHORS   Castillon GA, Michon L, Watanabe R
  TITLE     Apical sorting of lysoGPI-anchored proteins occurs independent of 
            association with detergent-resistant membranes but dependent on 
            their N-glycosylation.
  JOURNAL   Mol Biol Cell. 2013 Jun;24(12):2021-33. doi: 
            10.1091/mbc.E13-03-0160. Epub 2013 Apr 24.
  PUBMED    23615438
REFERENCE   2  (bases 1 to 6094)
  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: 
            "10.1091/mbc.E13-03-0160"; journalName: "Mol Biol Cell"; date: 
            "2013-06"; volume: "24"; issue: "12"; pages: "2021-33"
COMMENT     Sequence Label: pME-puro-CLIP-FLAG-CD59
FEATURES             Location/Qualifiers
     source          1..6094
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        25..524
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(60..83)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     457..476
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             552..1151
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(552..571)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1048..1068
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1683..1703)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(1688..2017,2018..2033,2034..2302)
                     /label=SR-alpha promoter
                     /note="hybrid promoter consisting of the SV40 early 
                     promoter plus part of the long terminal repeat of human 
                     T-lymphotrophic virus 1"
     rep_origin      1868..2003
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     LTR             2034..2302
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     intron          2332..2425
                     /label=SV40 intron
                     /note="modified SV40 intron with splice donor and acceptor 
                     sites"
     CDS             2558..3103
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylcytosine 
                     derivatives"
                     /label=CLIP-tag(TM)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             3113..3136
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3533..3667
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3570..3589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3624..3643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3706..3723)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3877..4465)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3957..3976)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4636..5427,5428..5496))
                     /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     5259..5278
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5497..5601)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5628..6083)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5765..5786)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5977..5996
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aagcttcgac ctcgaaattc taccgggtag gggaggcgct tttcccaagg cagtctggag
       61 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
      121 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
      181 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
      241 ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg
      301 agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt
      361 tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg
      421 gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca
      481 cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgacctgc atccatctag
      541 atcagcttac catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
      601 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
      661 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
      721 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
      781 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
      841 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
      901 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
      961 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     1021 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     1081 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     1141 ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc
     1201 atgcagggat gcagaaattg atgatctatt aaacaataaa gatgtccact aaaatggaag
     1261 tttttcctgt catactttgt taagaagggt gagaacagag tacctacatt ttgaatggaa
     1321 ggattggagc tacgggggtg ggggtggggt gggattagat aaatgcctgc tctttactga
     1381 aggctcttta ctattgcttt atgataatgt ttcatagttg gatatcataa tttaaacaag
     1441 caaaaccaaa ttaagggcca gctcattcct cccactcatg atctatagat ctatagatct
     1501 ctcgtgggat cattgttttt ctcttgattc ccactttgtg gttctaagta ctgtggtttc
     1561 caaatgtgtc agtttcatag cctgaagaac gagatcagca gcctctgttc cacatacact
     1621 tcattctcag tattgttttg ccaagttcta attccatcag aagctggtcg aagcttggct
     1681 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     1741 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     1801 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     1861 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     1921 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     1981 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcctccgat cgaggggctc
     2041 gcatctctcc ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc
     2101 gcgttctgcc gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt
     2161 taaagctcag gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca
     2221 gccggctctc cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt
     2281 ctgttctgcg ccgttacaga tccaagctct gaaaaaccag aaagttaact ggtaagttta
     2341 gtctttttgt cttttatttc aggtcccgga tccggtggtg caaatcaaag aactgctcct
     2401 cagtggatgt tgcctttact tctaggcctg tacggaagtg ttacttctgc tctaaaagct
     2461 gcggaattcc accatgggaa tccaaggagg gtctgtcctg ttcgggctgc tgctcgtcct
     2521 ggctgtcttc tgccattcag gtcatagcct gcagggcatg gacaaagact gcgaaatgaa
     2581 gcgcaccacc ctggatagcc ctctgggcaa gctggaactg tctgggtgcg aacagggcct
     2641 gcaccgtatc atcttcctgg gcaaaggaac atctgccgcc gacgccgtgg aagtgcctgc
     2701 cccagccgcc gtgctgggcg gaccagagcc actgatccag gccaccgcct ggctcaacgc
     2761 ctactttcac cagcctgagg ccatcgagga gttccctgtg ccagccctgc accacccagt
     2821 gttccagcag gagagcttta cccgccaggt gctgtggaaa ctgctgaaag tggtgaagtt
     2881 cggagaggtc atcagcgaga gccacctggc cgccctggtg ggcaatcccg ccgccaccgc
     2941 cgccgtgaac accgccctgg acggaaatcc cgtgcccatt ctgatcccct gccaccgggt
     3001 ggtgcagggc gacagcgacg tggggcccta cctgggcggg ctcgccgtga aagagtggct
     3061 gctggcccac gagggccaca gactgggcaa gcctgggctg ggtgccctgc aggactacaa
     3121 ggacgacgat gacaagcttc agtgctacaa ctgtcctaac ccaactgctg actgcaaaac
     3181 agccgtcaat tgttcatctg attttgatgc gtgtctcatt accaaagctg ggttacaagt
     3241 gtataacaag tgttggaagt ttgagcattg caatttcaac gacgtcacaa cccgcttgag
     3301 ggaaaatgag ctaacgtact actgctgcaa gaaggacctg tgtaacttta acgaacagct
     3361 cgagaatggt gggacatcct tatcagagaa aacagttctt ctgctggtga ctccatttct
     3421 ggcagcagcc tggagccttc atccctaagc ggccgctaga ctagtctaga gaaaaaacct
     3481 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     3541 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     3601 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     3661 ctggatcccc gggtaccgag ctcgaattaa ttcctcttcc gcttcctcgc tcactgactc
     3721 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3781 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3841 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3901 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3961 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4021 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     4081 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4141 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4201 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4261 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4321 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4381 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4441 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4501 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4561 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4621 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4681 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4741 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4801 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4861 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4921 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4981 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5041 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5101 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5161 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5221 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5281 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5341 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5401 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5461 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5521 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5581 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaaa ttgtaaacgt
     5641 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5701 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5761 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5821 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     5881 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     5941 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     6001 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     6061 taatgcgccg ctacagggcg cgtcccattc gcca
//
