LOCUS       sequence-109174-        5439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, T7 to an 
            HA-6xHIS epitope tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5439)
  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 5439)
  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: pTW094
FEATURES             Location/Qualifiers
     source          1..5439
                     /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..1005
                     /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"
     terminator      1026..1213
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1081..1105)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            complement(1333..2908)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1333..1530)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1536..2564)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1536..2564)
                     /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(2565..2908)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    3003..3036
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3045..3071
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3072..3089
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    3116..3172
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     promoter        complement(3179..3197)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3218..3234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3218..3234)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3231..3253)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3242..3258
                     /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(3266..3272,3273..3290,3291..3296))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3311..3332
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3449..3466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3620..4208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3700..3719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4379..5170,5171..5239))
                     /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     5002..5021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5240..5344)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5412..5430
                     /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 ccatggcaag catgactggt ggacagcaaa tgggtactag ttaacttcta
     1021 aataagcgaa tttcttatga tttatgattt ttattattaa ataagttata aaaaaaataa
     1081 gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta ttcttgagta
     1141 actctttcct gtaggtcagg ttgctttctc aggtatagta tgaggtcgct cttattgacc
     1201 acacctctac cggcagatcc gctagggata acagggtaag gatccgagat tgtactgaga
     1261 gtgcaccata atcatcgatg aattcgagct cgttttcgac actggatggc ggcgttagta
     1321 tcgaatcgac agcagtatag cgaccagcat tcacatacga ttgacgcatg atattacttt
     1381 ctgcgcactt aacttcgcat ctgggcagat gatgtcgagg cgaaaaaaaa tataaatcac
     1441 gctaacattt gattaaaata gaacaactac aatataaaaa aactatacaa atgacaagtt
     1501 cttgaaaaca agaatctttt tattgtcagt actgattatt cctttgccct cggacgagtg
     1561 ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc
     1621 gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg
     1681 tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag
     1741 agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc tgcaagctcc
     1801 ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag
     1861 aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg
     1921 accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg
     1981 aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg
     2041 acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca
     2101 atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg
     2161 ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatggc ctccgcgacc
     2221 ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca
     2281 cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag cacttccgga
     2341 atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg
     2401 gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga
     2461 gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga
     2521 aacttctcga cagacgtcgc ggtgagttca ggctttttac ccatggttgt ttatgttcgg
     2581 atgtgatgtg agaactgtat cctagcaaga ttttaaaagg aagtatatga aagaagaacc
     2641 tcagtggcaa atcctaacct tttatatttc tctacagggg cgcggcgtgg ggacaattca
     2701 acgcgtctgt gaggggagcg tttccctgct cgcaggtccg cagcgaggag ccgtaatttt
     2761 tgcttcgcgc cgtgcggcca tcaaaatgta tggatgcaaa tgattataca tggggatgta
     2821 tgggctaaat gtacgggcga cagtcacatc atgcccctga gctgcgcacg tcaagactgt
     2881 caaggagggt attctgggcc tccatgtcgc tggccgggtg acccggcggg gacgaggcaa
     2941 gctaaacaga tctggcgcgc cttaattaac cctatgcggt gtgaaatacc gcacagtcta
     3001 gaataacttc gtataatgta tgctatacga agttatcagg aacttaccca tacgatgttc
     3061 ctgactatgc gcaccatcat caccatcaca ctagttaatg tacataagcg gccgcgaatt
     3121 cgagctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc ttttgttccc
     3181 tttagtgagg gttaattccg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3241 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3301 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3361 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3421 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3481 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3541 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3601 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3661 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3721 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3781 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3841 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3901 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3961 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4021 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4081 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4141 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4201 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4261 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4321 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4381 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4441 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4501 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4561 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4621 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4681 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4741 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4801 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4861 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4921 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4981 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5041 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5101 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5161 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5221 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5281 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5341 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     5401 taacctataa aaataggcgt atcacgaggc cctttcgtc
//
