LOCUS       sequence-109173-        5435 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, HA-6xHIS to a T7
            epitope tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5435)
  AUTHORS   De Vos D, Frederiks F, Terweij M, van Welsem T, Verzijlbergen KF, 
            Iachina E, de Graaf EL, Altelaar AF, Oudgenoeg G, Heck AJ, 
            Krijgsveld J, Bakker BM, van Leeuwen F
  TITLE     Progressive methylation of ageing histones by Dot1 functions as a 
            timer.
  JOURNAL   EMBO Rep. 2011 Sep 1;12(9):956-62. doi: 10.1038/embor.2011.131.
  PUBMED    21760613
REFERENCE   2  (bases 1 to 5435)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/embor.2011"; journalName: "EMBO Rep"; date: "2011-09-1- 1";
            volume: "12"; issue: "9"; pages: "956-62"
COMMENT     Sequence Label: pTW087
FEATURES             Location/Qualifiers
     source          1..5435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(131..152)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     343..362
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     579..601
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     593..610
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     594..610
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     617..636
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        617..635
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    689..722
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             731..757
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             758..775
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      796..983
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(851..875)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            complement(1102..2677)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1102..1299)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1305..2333)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1305..2333)
                     /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(2334..2677)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    2772..2805
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2814..2846
                     /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(2932..2950)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2971..2987)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2971..2987)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2984..3006)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2995..3011
                     /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(3019..3025,3026..3043,3044..3049))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3064..3085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3202..3219)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3373..3961)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3453..3472)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4132..4923,4924..4992))
                     /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     4755..4774
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4993..5097)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5165..5183
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5221..5243)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5343..5362
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(join(5429..5435,1..449))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
       61 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
      121 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
      181 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
      241 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
      301 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
      361 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
      421 cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg ccattcaggc tgcgcaactg
      481 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
      541 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
      601 gacggccagt gaattgtaat acgactcact atagggcgaa ttgggcccaa tgcattggcg
      661 ccgcggccgc ggtggatctg gtggatctat aacttcgtat aatgtatgct atacgaagtt
      721 atcaggtacc tacccatacg atgttcctga ctatgcgcac catcatcacc atcacactag
      781 ttaacttcta aataagcgaa tttcttatga tttatgattt ttattattaa ataagttata
      841 aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta
      901 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagta tgaggtcgct
      961 cttattgacc acacctctac cggcagatcc gctagggata acagggtaag gatccgcatt
     1021 gtactgagag tgcaccataa tcatcgatga attcgagctc gttttcgaca ctggatggcg
     1081 gcgttagtat cgaatcgaca gcagtatagc gaccagcatt cacatacgat tgacgcatga
     1141 tattactttc tgcgcactta acttcgcatc tgggcagatg atgtcgaggc gaaaaaaaat
     1201 ataaatcacg ctaacatttg attaaaatag aacaactaca atataaaaaa actatacaaa
     1261 tgacaagttc ttgaaaacaa gaatcttttt attgtcagta ctgattattc ctttgccctc
     1321 ggacgagtgc tggggcgtcg gtttccacta tcggcgagta cttctacaca gccatcggtc
     1381 cagacggccg cgcttctgcg ggcgatttgt gtacgcccga cagtcccggc tccggatcgg
     1441 acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag
     1501 ctctgataga gttggtcaag accaatgcgg agcatatacg cccggagccg cggcgatcct
     1561 gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct gctccataca agccaaccac
     1621 ggcctccaga agaagatgtt ggcgacctcg tattgggaat ccccgaacat cgcctcgctc
     1681 cagtcaatga ccgctgttat gcggccattg tccgtcagga cattgttgga gccgaaatcc
     1741 gcgtgcacga ggtgccggac ttcggggcag tcctcggccc aaagcatcag ctcatcgaga
     1801 gcctgcgcga cggacgcact gacggtgtcg tccatcacag tttgccagtg atacacatgg
     1861 ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt attgaccgat tccttgcggt
     1921 ccgaatgggc cgaacccgct cgtctggcta agatcggccg cagcgatcgc atccatggcc
     1981 tccgcgaccg gctgcagaac agcgggcagt tcggtttcag gcaggtcttg caacgtgaca
     2041 ccctgtgcac ggcgggagat gcaataggtc aggctctcgc tgaattcccc aatgtcaagc
     2101 acttccggaa tcgggagcgc ggccgatgca aagtgccgat aaacataacg atctttgtag
     2161 aaaccatcgg cgcagctatt tacccgcagg acatatccac gccctcctac atcgaagctg
     2221 aaagcacgag attcttcgcc ctccgagagc tgcatcaggt cggagacgct gtcgaacttt
     2281 tcgatcagaa acttctcgac agacgtcgcg gtgagttcag gctttttacc catggttgtt
     2341 tatgttcgga tgtgatgtga gaactgtatc ctagcaagat tttaaaagga agtatatgaa
     2401 agaagaacct cagtggcaaa tcctaacctt ttatatttct ctacaggggc gcggcgtggg
     2461 gacaattcaa cgcgtctgtg aggggagcgt ttccctgctc gcaggtccgc agcgaggagc
     2521 cgtaattttt gcttcgcgcc gtgcggccat caaaatgtat ggatgcaaat gattatacat
     2581 ggggatgtat gggctaaatg tacgggcgac agtcacatca tgcccctgag ctgcgcacgt
     2641 caagactgtc aaggagggta ttctgggcct ccatgtcgct ggccgggtga cccggcgggg
     2701 acgaggcaag ctaaacagat ctggcgcgcc ttaattaacc ctatgcggtg tgaaataccg
     2761 cacagtctag aataacttcg tataatgtat gctatacgaa gttatcagtc gacatggcaa
     2821 gcatgactgg tggacagcaa atgggttgta cataagcggc cgcgaattca attcgagctc
     2881 ggtacccggg gatcctctag agtcgacctg caggcatgca agcttttgtt ccctttagtg
     2941 agggttaatt ccgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3001 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3061 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3121 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3181 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3241 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3301 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3361 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3421 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3481 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3541 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3601 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3661 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3721 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3781 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     3841 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3901 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3961 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4021 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4081 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4141 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4201 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4261 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4321 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4381 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4441 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4501 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4561 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4621 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4681 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4741 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4801 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4861 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4921 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4981 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5041 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5101 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     5161 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     5221 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     5281 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     5341 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     5401 atgcgtaagg agaaaatacc gcatcaggaa attgt
//
