LOCUS       sequence-109169-        5412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, HA to a T7 
            epitope tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5412)
  AUTHORS   Verzijlbergen KF, Menendez-Benito V, van Welsem T, van Deventer SJ, 
            Lindstrom DL, Ovaa H, Neefjes J, Gottschling DE, van Leeuwen F
  TITLE     Recombination-induced tag exchange to track old and new proteins.
  JOURNAL   Proc Natl Acad Sci U S A. 2010 Jan 5;107(1):64-8. doi: 
            10.1073/pnas.0911164107. Epub 2009 Dec 14.
  PUBMED    20018668
REFERENCE   2  (bases 1 to 5412)
  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: 
            "10.1073/pnas.0911164107"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2010-01-5- 5"; volume: "107"; issue: "1"; pages: "64-8"
COMMENT     Sequence Label: pFvL118
FEATURES             Location/Qualifiers
     source          1..5412
                     /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"
     terminator      1020..1207
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1075..1099)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            complement(1327..2902)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1327..1524)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1530..2558)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1530..2558)
                     /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(2559..2902)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    2997..3030
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3039..3071
                     /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"
     misc_feature    3089..3145
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     promoter        complement(3152..3170)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3191..3207)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3191..3207)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3204..3226)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3215..3231
                     /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(3239..3245,3246..3263,3264..3269))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3284..3305
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3422..3439)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3593..4181)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3673..3692)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4352..5143,5144..5212))
                     /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     4975..4994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5213..5317)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5385..5403
                     /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 ctagttaact tctaaataag
     1021 cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat
     1081 acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt
     1141 tcctgtaggt caggttgctt tctcaggtat agtatgaggt cgctcttatt gaccacacct
     1201 ctaccggcag atccgctagg gataacaggg taaggatccg agattgtact gagagtgcac
     1261 cataatcatc gatgaattcg agctcgtttt cgacactgga tggcggcgtt agtatcgaat
     1321 cgacagcagt atagcgacca gcattcacat acgattgacg catgatatta ctttctgcgc
     1381 acttaacttc gcatctgggc agatgatgtc gaggcgaaaa aaaatataaa tcacgctaac
     1441 atttgattaa aatagaacaa ctacaatata aaaaaactat acaaatgaca agttcttgaa
     1501 aacaagaatc tttttattgt cagtactgat tattcctttg ccctcggacg agtgctgggg
     1561 cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt
     1621 ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat
     1681 cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg
     1741 tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc
     1801 ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag
     1861 atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct
     1921 gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc
     1981 cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac
     2041 gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg
     2101 catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac
     2161 ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc
     2221 agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg
     2281 gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg
     2341 agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag
     2401 ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct
     2461 tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc
     2521 tcgacagacg tcgcggtgag ttcaggcttt ttacccatgg ttgtttatgt tcggatgtga
     2581 tgtgagaact gtatcctagc aagattttaa aaggaagtat atgaaagaag aacctcagtg
     2641 gcaaatccta accttttata tttctctaca ggggcgcggc gtggggacaa ttcaacgcgt
     2701 ctgtgagggg agcgtttccc tgctcgcagg tccgcagcga ggagccgtaa tttttgcttc
     2761 gcgccgtgcg gccatcaaaa tgtatggatg caaatgatta tacatgggga tgtatgggct
     2821 aaatgtacgg gcgacagtca catcatgccc ctgagctgcg cacgtcaaga ctgtcaagga
     2881 gggtattctg ggcctccatg tcgctggccg ggtgacccgg cggggacgag gcaagctaaa
     2941 cagatctggc gcgccttaat taaccctatg cggtgtgaaa taccgcacag tctagaataa
     3001 cttcgtataa tgtatgctat acgaagttat cagtcgacat ggcaagcatg actggtggac
     3061 agcaaatggg ttgtacataa gcggccgcga attcgagctc ggtacccggg gatcctctag
     3121 agtcgacctg caggcatgca agcttttgtt ccctttagtg agggttaatt ccgagcttgg
     3181 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3241 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3301 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3361 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3421 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3481 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3541 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3601 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3661 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3721 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3781 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3841 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3901 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3961 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4021 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4081 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4141 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4201 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4261 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4321 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4381 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4441 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4501 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4561 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4621 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4681 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4741 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4801 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4861 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4921 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4981 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5041 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5101 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5161 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5221 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5281 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5341 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     5401 ggccctttcg tc
//
