LOCUS       sequence-109176-        5444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, V5 to an 
            HA-6xHIS epitope tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5444)
  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 5444)
  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: pMT004
FEATURES             Location/Qualifiers
     source          1..5444
                     /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..1014
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     terminator      1032..1219
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1087..1111)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            complement(1338..2913)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1338..1535)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1541..2569)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1541..2569)
                     /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(2570..2913)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    3008..3041
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3050..3076
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3077..3094
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    3121..3177
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     promoter        complement(3184..3202)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3223..3239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3223..3239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3236..3258)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3247..3263
                     /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(3271..3277,3278..3295,3296..3301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3316..3337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3454..3471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3625..4213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3705..3724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4384..5175,5176..5244))
                     /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     5007..5026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5245..5349)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5417..5435
                     /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 ccggtaagcc tatccctaac cctctcctcg gtctcgattc tactagttaa
     1021 cttctaaata agcgaatttc ttatgattta tgatttttat tattaaataa gttataaaaa
     1081 aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa ttcttattct
     1141 tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagtatgag gtcgctctta
     1201 ttgaccacac ctctaccggc agatccgcta gggataacag ggtaaggatc cagattgtac
     1261 tgagagtgca ccataatcat cgatgaattc gagctcgttt tcgacactgg atggcggcgt
     1321 tagtatcgaa tcgacagcag tatagcgacc agcattcaca tacgattgac gcatgatatt
     1381 actttctgcg cacttaactt cgcatctggg cagatgatgt cgaggcgaaa aaaaatataa
     1441 atcacgctaa catttgatta aaatagaaca actacaatat aaaaaaacta tacaaatgac
     1501 aagttcttga aaacaagaat ctttttattg tcagtactga ttattccttt gccctcggac
     1561 gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga
     1621 cggccgcgct tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga
     1681 ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct
     1741 gatagagttg gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa
     1801 gctccggatg cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc
     1861 tccagaagaa gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt
     1921 caatgaccgc tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt
     1981 gcacgaggtg ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct
     2041 gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat
     2101 cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga
     2161 atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg
     2221 cgaccggctg cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct
     2281 gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt
     2341 ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac
     2401 catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag
     2461 cacgagattc ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga
     2521 tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttacccatg gttgtttatg
     2581 ttcggatgtg atgtgagaac tgtatcctag caagatttta aaaggaagta tatgaaagaa
     2641 gaacctcagt ggcaaatcct aaccttttat atttctctac aggggcgcgg cgtggggaca
     2701 attcaacgcg tctgtgaggg gagcgtttcc ctgctcgcag gtccgcagcg aggagccgta
     2761 atttttgctt cgcgccgtgc ggccatcaaa atgtatggat gcaaatgatt atacatgggg
     2821 atgtatgggc taaatgtacg ggcgacagtc acatcatgcc cctgagctgc gcacgtcaag
     2881 actgtcaagg agggtattct gggcctccat gtcgctggcc gggtgacccg gcggggacga
     2941 ggcaagctaa acagatctgg cgcgccttaa ttaaccctat gcggtgtgaa ataccgcaca
     3001 gtctagaata acttcgtata atgtatgcta tacgaagtta tcaggaactt acccatacga
     3061 tgttcctgac tatgcgcacc atcatcacca tcacactagt taatgtacat aagcggccgc
     3121 gaattcgagc tcggtacccg gggatcctct agagtcgacc tgcaggcatg caagcttttg
     3181 ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt
     3241 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3301 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3361 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3421 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3481 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3541 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3601 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3661 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3721 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3781 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3841 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3901 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3961 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4021 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     4081 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4141 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4201 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4261 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4321 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4381 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4441 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4501 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4561 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4621 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4681 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4741 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4801 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4861 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4921 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4981 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5041 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5101 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5161 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5221 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5281 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5341 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     5401 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
