LOCUS       sequence-109182-        6162 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, 3xHA to 
            3xT7-mRFP tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6162)
  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 6162)
  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: pKV014
FEATURES             Location/Qualifiers
     source          1..6162
                     /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..999
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1006..1032
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1036..1062
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      1083..1270
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1138..1162)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            complement(1389..2964)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1389..1586)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1592..2620)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1592..2620)
                     /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(2621..2964)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    3059..3092
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3101..3133
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             3215..3889
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3215..3889
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(3345..3363)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3613..3632
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3807..3830
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     promoter        complement(3902..3920)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3941..3957)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3941..3957)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3954..3976)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3965..3981
                     /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(3989..3995,3996..4013,4014..4019))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4034..4055
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4172..4189)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4343..4931)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4423..4442)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5102..5893,5894..5962))
                     /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     5725..5744
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5963..6067)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6135..6153
                     /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 cctacccata cgatgttcct gactatgcga ctagctatcc ctatgacgtc
     1021 ccggactatg caggatatcc atatgacgtt ccagattacg ctactagtta acttctaaat
     1081 aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa aaaataagtg
     1141 tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc ttgagtaact
     1201 ctttcctgta ggtcaggttg ctttctcagg tatagtatga ggtcgctctt attgaccaca
     1261 cctctaccgg cagatccgct agggataaca gggtaaggat ccagattgta ctgagagtgc
     1321 accataatca tcgatgaatt cgagctcgtt ttcgacactg gatggcggcg ttagtatcga
     1381 atcgacagca gtatagcgac cagcattcac atacgattga cgcatgatat tactttctgc
     1441 gcacttaact tcgcatctgg gcagatgatg tcgaggcgaa aaaaaatata aatcacgcta
     1501 acatttgatt aaaatagaac aactacaata taaaaaaact atacaaatga caagttcttg
     1561 aaaacaagaa tctttttatt gtcagtactg attattcctt tgccctcgga cgagtgctgg
     1621 ggcgtcggtt tccactatcg gcgagtactt ctacacagcc atcggtccag acggccgcgc
     1681 ttctgcgggc gatttgtgta cgcccgacag tcccggctcc ggatcggacg attgcgtcgc
     1741 atcgaccctg cgcccaagct gcatcatcga aattgccgtc aaccaagctc tgatagagtt
     1801 ggtcaagacc aatgcggagc atatacgccc ggagccgcgg cgatcctgca agctccggat
     1861 gcctccgctc gaagtagcgc gtctgctgct ccatacaagc caaccacggc ctccagaaga
     1921 agatgttggc gacctcgtat tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg
     1981 ctgttatgcg gccattgtcc gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt
     2041 gccggacttc ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg
     2101 acgcactgac ggtgtcgtcc atcacagttt gccagtgata cacatgggga tcagcaatcg
     2161 cgcatatgaa atcacgccat gtagtgtatt gaccgattcc ttgcggtccg aatgggccga
     2221 acccgctcgt ctggctaaga tcggccgcag cgatcgcatc catggcctcc gcgaccggct
     2281 gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc
     2341 gggagatgca ataggtcagg ctctcgctga attccccaat gtcaagcact tccggaatcg
     2401 ggagcgcggc cgatgcaaag tgccgataaa cataacgatc tttgtagaaa ccatcggcgc
     2461 agctatttac ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa gcacgagatt
     2521 cttcgccctc cgagagctgc atcaggtcgg agacgctgtc gaacttttcg atcagaaact
     2581 tctcgacaga cgtcgcggtg agttcaggct ttttacccat ggttgtttat gttcggatgt
     2641 gatgtgagaa ctgtatccta gcaagatttt aaaaggaagt atatgaaaga agaacctcag
     2701 tggcaaatcc taacctttta tatttctcta caggggcgcg gcgtggggac aattcaacgc
     2761 gtctgtgagg ggagcgtttc cctgctcgca ggtccgcagc gaggagccgt aatttttgct
     2821 tcgcgccgtg cggccatcaa aatgtatgga tgcaaatgat tatacatggg gatgtatggg
     2881 ctaaatgtac gggcgacagt cacatcatgc ccctgagctg cgcacgtcaa gactgtcaag
     2941 gagggtattc tgggcctcca tgtcgctggc cgggtgaccc ggcggggacg aggcaagcta
     3001 aacagatctg gcgcgcctta attaacccta tgcggtgtga aataccgcac agtctagaat
     3061 aacttcgtat aatgtatgct atacgaagtt atcagtcgac atggcaagca tgactggtgg
     3121 acagcaaatg ggttgtacta tggcttctat gacaggaggt caacagatgg gaggaatggc
     3181 ctcaatgacc ggtggccagc aaatgggatg tacaatggcc tcctccgagg acgtcatcaa
     3241 ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg aacggccacg agttcgagat
     3301 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     3361 caagggcggc cccctgccct tcgcctggga catcctgtcc cctcagttcc agtacggctc
     3421 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     3481 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     3541 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
     3601 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg
     3661 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag atgaggctga agctgaagga
     3721 cggcggccac tacgacgccg aggtcaagac cacctacatg gccaagaagc ccgtgcagct
     3781 gcccggcgcc tacaagaccg acatcaagct ggacatcacc tcccacaacg aggactacac
     3841 catcgtggaa cagtacgagc gcgccgaggg ccgccactcc accggcgcca agcttttgtt
     3901 ccctttagtg agggttaatt tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3961 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4021 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4081 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4141 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4201 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4261 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4321 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4381 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4441 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4501 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4561 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4621 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4681 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4741 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4801 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4861 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4921 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4981 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5041 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5101 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5161 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5221 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5281 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5341 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5401 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5461 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5521 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5581 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5641 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5701 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5761 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5821 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5881 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5941 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6001 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6061 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     6121 cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
