LOCUS       sequence-109181-        5421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, HA to a T7 
            epitope tag, invariable V5 tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5421)
  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 5421)
  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: pKV006
FEATURES             Location/Qualifiers
     source          1..5421
                     /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"
     CDS             938..979
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    986..1019
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1028..1054
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      1075..1262
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1130..1154)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            complement(1381..2956)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1381..1578)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1584..2612)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1584..2612)
                     /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(2613..2956)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    3051..3084
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3093..3125
                     /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"
     promoter        complement(3161..3179)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3200..3216)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3200..3216)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3213..3235)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3224..3240
                     /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(3248..3254,3255..3272,3273..3278))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3293..3314
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3431..3448)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3602..4190)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3682..3701)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4361..5152,5153..5221))
                     /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     4984..5003
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5222..5326)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5394..5412
                     /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 tgctagcggt aagcctatcc ctaaccctct
      961 cctcggtctc gattctacga gcgctataac ttcgtataat gtatgctata cgaagttatc
     1021 aggtacctac ccatacgatg ttcctgacta tgcgactagt taacttctaa ataagcgaat
     1081 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa
     1141 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg
     1201 taggtcaggt tgctttctca ggtatagtat gaggtcgctc ttattgacca cacctctacc
     1261 ggcagatccg ctagggataa cagggtaagg atccagattg tactgagagt gcaccataat
     1321 catcgatgaa ttcgagctcg ttttcgacac tggatggcgg cgttagtatc gaatcgacag
     1381 cagtatagcg accagcattc acatacgatt gacgcatgat attactttct gcgcacttaa
     1441 cttcgcatct gggcagatga tgtcgaggcg aaaaaaaata taaatcacgc taacatttga
     1501 ttaaaataga acaactacaa tataaaaaaa ctatacaaat gacaagttct tgaaaacaag
     1561 aatcttttta ttgtcagtac tgattattcc tttgccctcg gacgagtgct ggggcgtcgg
     1621 tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc gcttctgcgg
     1681 gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc gcatcgaccc
     1741 tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag ttggtcaaga
     1801 ccaatgcgga gcatatacgc ccggagccgc ggcgatcctg caagctccgg atgcctccgc
     1861 tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa gaagatgttg
     1921 gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac cgctgttatg
     1981 cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag gtgccggact
     2041 tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac ggacgcactg
     2101 acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat cgcgcatatg
     2161 aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc
     2221 gtctggctaa gatcggccgc agcgatcgca tccatggcct ccgcgaccgg ctgcagaaca
     2281 gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg gcgggagatg
     2341 caataggtca ggctctcgct gaattcccca atgtcaagca cttccggaat cgggagcgcg
     2401 gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc gcagctattt
     2461 acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc
     2521 tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa cttctcgaca
     2581 gacgtcgcgg tgagttcagg ctttttaccc atggttgttt atgttcggat gtgatgtgag
     2641 aactgtatcc tagcaagatt ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaat
     2701 cctaaccttt tatatttctc tacaggggcg cggcgtgggg acaattcaac gcgtctgtga
     2761 ggggagcgtt tccctgctcg caggtccgca gcgaggagcc gtaatttttg cttcgcgccg
     2821 tgcggccatc aaaatgtatg gatgcaaatg attatacatg gggatgtatg ggctaaatgt
     2881 acgggcgaca gtcacatcat gcccctgagc tgcgcacgtc aagactgtca aggagggtat
     2941 tctgggcctc catgtcgctg gccgggtgac ccggcgggga cgaggcaagc taaacagatc
     3001 tggcgcgcct taattaaccc tatgcggtgt gaaataccgc acagtctaga ataacttcgt
     3061 ataatgtatg ctatacgaag ttatcagtcg acatggcaag catgactggt ggacagcaaa
     3121 tgggttgtac ataagcggcc gcgcatgcaa gcttttgttc cctttagtga gggttaattt
     3181 cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3241 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3301 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3361 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3421 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3481 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3541 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3601 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3661 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3721 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3781 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3841 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3901 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3961 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4021 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4081 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4141 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4201 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4261 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4321 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4381 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4441 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4501 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4561 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4621 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4681 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4741 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4801 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4861 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4921 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4981 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5041 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5101 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5161 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5221 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5281 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5341 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5401 gtatcacgag gccctttcgt c
//
