LOCUS       sequence-112999-        5382 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, HA to a 2xFLAG 
            epitope tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5382)
  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 5382)
  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: pMT010
FEATURES             Location/Qualifiers
     source          1..5382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(237..692)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(374..395)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     586..605
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     822..844
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     836..853
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     837..853
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     860..879
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        860..878
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    932..965
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             974..1000
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      1021..1208
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1076..1100)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            complement(1327..2902)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1327..1524)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1530..2558)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1530..2558)
                     /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(2559..2902)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    2997..3030
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3039..3086
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     promoter        complement(3122..3140)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3161..3177)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3161..3177)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3174..3196)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3185..3201
                     /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(3209..3215,3216..3233,3234..3239))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3254..3275
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3392..3409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3563..4151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3643..3662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4322..5113,5114..5182))
                     /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     4945..4964
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5183..5287)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5355..5373
                     /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 gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat
      241 tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
      301 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
      361 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
      421 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
      481 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
      541 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
      601 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
      661 cgccgcgctt aatgcgccgc tacagggcgc gtcgcgccat tcgccattca ggctgcgcaa
      721 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
      781 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
      841 aacgacggcc agtgaattgt aatacgactc actatagggc gaattgggcc caatgcattg
      901 gcgccgcggc cgcggtggat ctggtggatc tataacttcg tataatgtat gctatacgaa
      961 gttatcaggt acctacccat acgatgttcc tgactatgcg actagttaac ttctaaataa
     1021 gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta
     1081 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct
     1141 ttcctgtagg tcaggttgct ttctcaggta tagtatgagg tcgctcttat tgaccacacc
     1201 tctaccggca gatccgctag ggataacagg gtaaggatcc agattgtact gagagtgcac
     1261 cataatcatc gatgaattcg agctcgtttt cgacactgga tggcggcgtt agtatcgaat
     1321 cgacagcagt atagcgacca gcattcacat acgattgacg catgatatta ctttctgcgc
     1381 acttaacttc gcatctgggc agatgatgtc gaggcgaaaa aaaatataaa tcacgctaac
     1441 atttgattaa aatagaacaa ctacaatata aaaaaactat acaaatgaca agttcttgaa
     1501 aacaagaatc tttttattgt cagtactgat tattcctttg ccctcggacg agtgctgggg
     1561 cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt
     1621 ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat
     1681 cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg
     1741 tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc
     1801 ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag
     1861 atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct
     1921 gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc
     1981 cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac
     2041 gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg
     2101 catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac
     2161 ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc
     2221 agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg
     2281 gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg
     2341 agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag
     2401 ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct
     2461 tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc
     2521 tcgacagacg tcgcggtgag ttcaggcttt ttacccatgg ttgtttatgt tcggatgtga
     2581 tgtgagaact gtatcctagc aagattttaa aaggaagtat atgaaagaag aacctcagtg
     2641 gcaaatccta accttttata tttctctaca ggggcgcggc gtggggacaa ttcaacgcgt
     2701 ctgtgagggg agcgtttccc tgctcgcagg tctgcagcga ggagccgtaa tttttgcttc
     2761 gcgccgtgcg gccatcaaaa tgtatggatg caaatgatta tacatgggga tgtatgggct
     2821 aaatgtacgg gcgacagtca catcatgccc ctgagctgcg cacgtcaaga ctgtcaagga
     2881 gggtattctg ggcctccatg tcgctggccg ggtgacccgg cggggacgag gcaagctaaa
     2941 cagatctggc gcgccttaat taaccctatg cggtgtgaaa taccgcacag tctagaataa
     3001 cttcgtataa tgtatgctat acgaagttat cagtcgacga ctacaaggac gatgacgata
     3061 aagattataa agatgacgat gacaagtgta cataagcggc cgcgcatgca agcttttgtt
     3121 ccctttagtg agggttaatt tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3181 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3241 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3301 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3361 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3421 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3481 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3541 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3601 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3661 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3721 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3781 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3841 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3901 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3961 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4021 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4081 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4141 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4201 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4261 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4321 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4381 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4441 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4501 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4561 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4621 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4681 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4741 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4801 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4861 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4921 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4981 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5041 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5101 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5161 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5221 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5281 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     5341 cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
