LOCUS       sequence-109172-        5417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, T7 to an HA 
            epitope tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5417)
  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 5417)
  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: pTW081
FEATURES             Location/Qualifiers
     source          1..5417
                     /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"
     promoter        complement(3157..3175)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3196..3212)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3196..3212)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3209..3231)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3220..3236
                     /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(3244..3250,3251..3268,3269..3274))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3289..3310
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3427..3444)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3598..4186)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3678..3697)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4357..5148,5149..5217))
                     /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     4980..4999
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5218..5322)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5390..5408
                     /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 agttatcagt cgactaccca tacgatgttc
     3061 ctgactatgc gtgtacataa gcggccgcga attcaattcg agctcggtac ccggggatcc
     3121 tctagagtcg acctgcaggc atgcaagctt ttgttccctt tagtgagggt taattccgag
     3181 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3241 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3301 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3361 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3421 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3481 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3541 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3601 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3661 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3721 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3781 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3841 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3901 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3961 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4021 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4081 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4141 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4201 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4261 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4321 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4381 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4441 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4501 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4561 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4621 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4681 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4741 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4801 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4861 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4921 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4981 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5041 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5101 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5161 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5221 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5281 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5341 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     5401 cacgaggccc tttcgtc
//
