LOCUS       sequence-311423-        4606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For C-terminal 3xHA-3xFLAG-protein tagging in S. pombe using hphMX4 
            as resistance marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4606)
  AUTHORS   Kilchert C, Kecman T, Priest E, Hester S, Aydin E, Kus K, Rossbach 
            O, Castello A, Mohammed S, Vasiljeva L
  TITLE     System-wide analyses of the fission yeast poly(A)(+) RNA interactome
            reveal insights into organization and function of RNA-protein 
            complexes.
  JOURNAL   Genome Res. 2020 Jul;30(7):1012-1026. doi: 10.1101/gr.257006.119. 
            Epub 2020 Jun 18.
  PUBMED    32554781
REFERENCE   2  (bases 1 to 4606)
  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: 
            "10.1101/gr.257006.119"; journalName: "Genome Res"; date: "2020-07";
            volume: "30"; issue: "7"; pages: "1012-1026"
COMMENT     Sequence Label: pAG32-3HA-3FLAG
FEATURES             Location/Qualifiers
     source          1..4606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..72
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        153..171
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     153..170
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             235..324
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             355..420
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      468..655
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(523..547)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            730..2305
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        730..1073
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1074..2102
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1074..2102
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2108..2305
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2412..2431)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2413..2431)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2518..2535)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2689..3277)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2769..2788)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3448..4239,4240..4308))
                     /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     4071..4090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4309..4413)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4481..4499
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4537..4559)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
       61 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat
      121 taatacataa ccttatgtat catacacata cgatttaggt gacactatag aacgcggccg
      181 ccagctgaag cttcgtacgc tgcaggtcga cggatccccg ggttaattaa cttttaccca
      241 tacgatgttc ctgactatgc gggctatccg tatgacgtcc cggactatgc aggttcctat
      301 ccatatgacg ttccagatta cgctattaac atggaagggg gaggcggggg tggagactac
      361 aaagaccatg acggtgatta taaagatcat gacatcgact acaaggatga cgatgacaag
      421 tagagcttga tatcgaattc ctgcagcccc gcgccacttc taaataagcg aatttcttat
      481 gatttatgat ttttattatt aaataagtta taaaaaaaat aagtgtatac aaattttaaa
      541 gtgactctta ggttttaaaa cgaaaattct tattcttgag taactctttc ctgtaggtca
      601 ggttgctttc tcaggtatag tatgaggtcg ctcttattga ccacacctct accggcagat
      661 ccgctaggga taacagggta atatagatct gtttagcttg cctcgtcccc gccgggtcac
      721 ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca
      781 ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt
      841 gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcgg
      901 acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc
      961 ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct
     1021 tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca accatgggta
     1081 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     1141 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     1201 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     1261 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     1321 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     1381 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     1441 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     1501 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     1561 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     1621 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     1681 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     1741 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     1801 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     1861 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     1921 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     1981 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     2041 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     2101 aatcagtact gacaataaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt
     2161 ttatattgta gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct
     2221 cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg
     2281 tatgtgaatg ctggtcgcta tactgctgtc gattcgatac taacgccgcc atccagtgtc
     2341 gaaaacgagc tcgaattcat cgatgatatc agatccacta gtggcctatg cggccgcgga
     2401 tctgccggtc tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta
     2461 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2521 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2581 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2641 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2701 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2761 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2821 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2881 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2941 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3001 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3061 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3121 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3181 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3241 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3301 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3361 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3421 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3481 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3541 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     3601 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3661 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3721 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     3781 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3841 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3901 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3961 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4021 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4081 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4141 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4201 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4261 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4321 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4381 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4441 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     4501 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     4561 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcc
//
