LOCUS       sequence-109171-        5414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, HA to a V5 
            epitope tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5414)
  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 5414)
  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: pTW073
FEATURES             Location/Qualifiers
     source          1..5414
                     /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(1328..2903)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1328..1525)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1531..2559)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1531..2559)
                     /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(2560..2903)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    2998..3031
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3154..3172)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3193..3209)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3193..3209)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3206..3228)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3217..3233
                     /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(3241..3247,3248..3265,3266..3271))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3286..3307
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3424..3441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3595..4183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3675..3694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4354..5145,5146..5214))
                     /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     4977..4996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5215..5319)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5387..5405
                     /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 gagattgtac tgagagtgca
     1261 ccataatcat cgatgaattc gagctcgttt tcgacactgg atggcggcgt tagtatcgaa
     1321 tcgacagcag tatagcgacc agcattcaca tacgattgac gcatgatatt actttctgcg
     1381 cacttaactt cgcatctggg cagatgatgt cgaggcgaaa aaaaatataa atcacgctaa
     1441 catttgatta aaatagaaca actacaatat aaaaaaacta tacaaatgac aagttcttga
     1501 aaacaagaat ctttttattg tcagtactga ttattccttt gccctcggac gagtgctggg
     1561 gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct
     1621 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca
     1681 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg
     1741 gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg
     1801 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa
     1861 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
     1921 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
     1981 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga
     2041 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc
     2101 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa
     2161 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg
     2221 cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
     2281 ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
     2341 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca
     2401 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc
     2461 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
     2521 ctcgacagac gtcgcggtga gttcaggctt tttacccatg gttgtttatg ttcggatgtg
     2581 atgtgagaac tgtatcctag caagatttta aaaggaagta tatgaaagaa gaacctcagt
     2641 ggcaaatcct aaccttttat atttctctac aggggcgcgg cgtggggaca attcaacgcg
     2701 tctgtgaggg gagcgtttcc ctgctcgcag gtccgcagcg aggagccgta atttttgctt
     2761 cgcgccgtgc ggccatcaaa atgtatggat gcaaatgatt atacatgggg atgtatgggc
     2821 taaatgtacg ggcgacagtc acatcatgcc cctgagctgc gcacgtcaag actgtcaagg
     2881 agggtattct gggcctccat gtcgctggcc gggtgacccg gcggggacga ggcaagctaa
     2941 acagatctgg cgcgccttaa ttaaccctat gcggtgtgaa ataccgcaca gtctagaata
     3001 acttcgtata atgtatgcta tacgaagtta tcagtcgacg gtaagcctat ccctaaccct
     3061 ctcctcggtc tcgattcttg tacataagcg gccgcggagc tcggtacccg gggatcctct
     3121 agagtcgacc tgcaggcatg caagcttttg ttccctttag tgagggttaa ttccgagctt
     3181 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3241 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3301 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3361 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3421 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3481 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3541 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3601 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3661 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3721 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3781 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3841 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3901 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3961 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4021 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4081 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4141 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4201 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4261 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4321 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4381 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4441 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4501 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4561 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4621 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4681 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4741 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4801 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4861 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4921 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4981 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5041 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5101 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5161 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5221 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5281 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5341 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     5401 gaggcccttt cgtc
//
