LOCUS       sequence-109178-        5453 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, 2xFLAG to an 
            HA-6xHIS epitope tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5453)
  AUTHORS   Terweij M, van Welsem T, van Deventer S, Verzijlbergen KF, 
            Menendez-Benito V, Ontoso D, San-Segundo P, Neefjes J, van Leeuwen F
  TITLE     Recombination-induced tag exchange (RITE) cassette series to monitor
            protein dynamics in Saccharomyces cerevisiae.
  JOURNAL   G3 (Bethesda). 2013 Aug 7;3(8):1261-72. doi: 10.1534/g3.113.006213.
  PUBMED    23708297
REFERENCE   2  (bases 1 to 5453)
  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: "10.1534/g3.113"; 
            journalName: "G3 (Bethesda)"; date: "2013-08-7- 7"; volume: "3"; 
            issue: "8"; pages: "1261-72"
COMMENT     Sequence Label: pMT006
FEATURES             Location/Qualifiers
     source          1..5453
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      complement(236..691)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(373..394)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..604
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     821..843
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     835..852
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     836..852
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     859..878
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        859..877
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    931..964
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             973..1020
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     terminator      1041..1228
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1096..1120)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            complement(1347..2922)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1347..1544)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1550..2578)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1550..2578)
                     /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"
     promoter        complement(2579..2922)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    3017..3050
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3059..3085
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3086..3103
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    3130..3186
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     promoter        complement(3193..3211)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3232..3248)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3232..3248)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3245..3267)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3256..3272
                     /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(3280..3286,3287..3304,3305..3310))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3325..3346
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3463..3480)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3634..4222)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3714..3733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4393..5184,5185..5253))
                     /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     5016..5035
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5254..5358)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5426..5444
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gccattgtac tgagagtgca
      181 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
      241 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
      301 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
      361 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
      421 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
      481 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
      541 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
      601 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
      661 gccgcgctta atgcgccgct acagggcgcg tcgcgccatt cgccattcag gctgcgcaac
      721 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
      781 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
      841 acgacggcca gtgaattgta atacgactca ctatagggcg aattgggccc aatgcattgg
      901 cgccgcggcc gcggtggatc tggtggatct ataacttcgt ataatgtatg ctatacgaag
      961 ttatcaggta ccgactacaa ggacgatgac gataaagatt ataaagatga cgatgacaag
     1021 actagttaac ttctaaataa gcgaatttct tatgatttat gatttttatt attaaataag
     1081 ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat
     1141 tcttattctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagtatgagg
     1201 tcgctcttat tgaccacacc tctaccggca gatccgctag ggataacagg gtaaggatcc
     1261 agattgtact gagagtgcac cataatcatc gatgaattcg agctcgtttt cgacactgga
     1321 tggcggcgtt agtatcgaat cgacagcagt atagcgacca gcattcacat acgattgacg
     1381 catgatatta ctttctgcgc acttaacttc gcatctgggc agatgatgtc gaggcgaaaa
     1441 aaaatataaa tcacgctaac atttgattaa aatagaacaa ctacaatata aaaaaactat
     1501 acaaatgaca agttcttgaa aacaagaatc tttttattgt cagtactgat tattcctttg
     1561 ccctcggacg agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat
     1621 cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg
     1681 atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa
     1741 ccaagctctg atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg
     1801 atcctgcaag ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca
     1861 accacggcct ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct
     1921 cgctccagtc aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga
     1981 aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat
     2041 cgagagcctg cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca
     2101 catggggatc agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt
     2161 gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca
     2221 tggcctccgc gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg
     2281 tgacaccctg tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt
     2341 caagcacttc cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt
     2401 tgtagaaacc atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga
     2461 agctgaaagc acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga
     2521 acttttcgat cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt ttacccatgg
     2581 ttgtttatgt tcggatgtga tgtgagaact gtatcctagc aagattttaa aaggaagtat
     2641 atgaaagaag aacctcagtg gcaaatccta accttttata tttctctaca ggggcgcggc
     2701 gtggggacaa ttcaacgcgt ctgtgagggg agcgtttccc tgctcgcagg tccgcagcga
     2761 ggagccgtaa tttttgcttc gcgccgtgcg gccatcaaaa tgtatggatg caaatgatta
     2821 tacatgggga tgtatgggct aaatgtacgg gcgacagtca catcatgccc ctgagctgcg
     2881 cacgtcaaga ctgtcaagga gggtattctg ggcctccatg tcgctggccg ggtgacccgg
     2941 cggggacgag gcaagctaaa cagatctggc gcgccttaat taaccctatg cggtgtgaaa
     3001 taccgcacag tctagaataa cttcgtataa tgtatgctat acgaagttat caggaactta
     3061 cccatacgat gttcctgact atgcgcacca tcatcaccat cacactagtt aatgtacata
     3121 agcggccgcg aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc
     3181 aagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct
     3241 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3301 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3361 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3421 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3481 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3541 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3601 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3661 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3721 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3781 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3841 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3901 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3961 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4021 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     4081 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4141 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4201 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4261 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4321 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4381 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4441 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4501 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4561 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4621 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4681 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4741 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4801 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4861 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4921 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4981 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5041 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5101 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5161 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5221 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5281 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5341 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     5401 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtc
//
