LOCUS       sequence-109185-        6069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination-Induced Tag Exchange (RITE) Cassette, T7 to an HA-GFP 
            tag, Hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6069)
  AUTHORS   Menendez-Benito V, van Deventer SJ, Jimenez-Garcia V, Roy-Luzarraga 
            M, van Leeuwen F, Neefjes J
  TITLE     Spatiotemporal analysis of organelle and macromolecular complex 
            inheritance.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Jan 2;110(1):175-80. doi: 
            10.1073/pnas.1207424110. Epub 2012 Dec 17.
  PUBMED    23248297
REFERENCE   2  (bases 1 to 6069)
  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.1207424110"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2013-01-2- 2"; volume: "110"; issue: "1"; pages: "175-80"
COMMENT     Sequence Label: pVM013
FEATURES             Location/Qualifiers
     source          1..6069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(15..470)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(152..173)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     364..383
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     600..622
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     614..631
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     615..631
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     638..657
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        638..656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    710..743
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             752..784
                     /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      805..992
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(860..884)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            complement(1111..2686)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1111..1308)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1314..2342)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1314..2342)
                     /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(2343..2686)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    2781..2814
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2823..2849
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2856..3572
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2856..3569
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        complement(3587..3605)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3626..3642)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3626..3642)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3639..3661)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3650..3666
                     /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(3674..3680,3681..3698,3699..3704))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3719..3740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3857..3874)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4028..4616)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4108..4127)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4787..5578,5579..5647))
                     /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     5410..5429
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5648..5752)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5820..5838
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5876..5898)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5998..6017
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aataccgcat caggaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg
       61 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
      121 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
      181 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
      241 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
      301 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
      361 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
      421 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cgcgccattc
      481 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
      541 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
      601 ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga
      661 attgggccca atgcattggc gccgcggccg cggtggatct ggtggatcta taacttcgta
      721 taatgtatgc tatacgaagt tatcaggtac catggcaagc atgactggtg gacagcaaat
      781 gggtactagt taacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa
      841 taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga
      901 aaattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagtat
      961 gaggtcgctc ttattgacca cacctctacc ggcagatccg ctagggataa cagggtaagg
     1021 atccgcattg tactgagagt gcaccataat catcgatgaa ttcgagctcg ttttcgacac
     1081 tggatggcgg cgttagtatc gaatcgacag cagtatagcg accagcattc acatacgatt
     1141 gacgcatgat attactttct gcgcacttaa cttcgcatct gggcagatga tgtcgaggcg
     1201 aaaaaaaata taaatcacgc taacatttga ttaaaataga acaactacaa tataaaaaaa
     1261 ctatacaaat gacaagttct tgaaaacaag aatcttttta ttgtcagtac tgattattcc
     1321 tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac ttctacacag
     1381 ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac agtcccggct
     1441 ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg
     1501 tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc ccggagccgc
     1561 ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa
     1621 gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc cccgaacatc
     1681 gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac attgttggag
     1741 ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca aagcatcagc
     1801 tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt ttgccagtga
     1861 tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt
     1921 ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc agcgatcgca
     1981 tccatggcct ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg caggtcttgc
     2041 aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct gaattcccca
     2101 atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata aacataacga
     2161 tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg ccctcctaca
     2221 tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc ggagacgctg
     2281 tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg ctttttaccc
     2341 atggttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt ttaaaaggaa
     2401 gtatatgaaa gaagaacctc agtggcaaat cctaaccttt tatatttctc tacaggggcg
     2461 cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg caggtccgca
     2521 gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg gatgcaaatg
     2581 attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat gcccctgagc
     2641 tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtcgctg gccgggtgac
     2701 ccggcgggga cgaggcaagc taaacagatc tggcgcgcct taattaaccc tatgcggtgt
     2761 gaaataccgc acagtctaga ataacttcgt ataatgtatg ctatacgaag ttatcagtcg
     2821 actacccata cgatgttcct gactatgcgt gtacaatgtc taaaggtgaa gaattattca
     2881 ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac aaattttctg
     2941 tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa tttatttgta
     3001 ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaact tatggtgttc
     3061 aatgtttttc tagataccca gatcatatga aacaacatga ctttttcaag tctgccatgc
     3121 cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac tacaagacca
     3181 gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta aaaggtattg
     3241 attttaaaga agatggtaac attttaggtc acaaattgga atacaactat aactctcaca
     3301 atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc aaaattagac
     3361 acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat actccaattg
     3421 gtgatggtcc agtcttgtta ccagacaacc attacttatc cactcaatct gccttatcca
     3481 aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact gctgctggta
     3541 ttacccatgg tatggatgaa ttgtataaat aaaaaagctt ttgttccctt tagtgagggt
     3601 taattccgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3661 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3721 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3781 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3841 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3901 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3961 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4021 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4081 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4141 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4201 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4261 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4321 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4381 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4441 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4501 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4561 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4621 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4681 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4741 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4801 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4861 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4921 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4981 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5041 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5101 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5161 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5221 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5281 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5341 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5401 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5461 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5521 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5581 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5641 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5701 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5761 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     5821 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     5881 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     5941 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     6001 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
     6061 taaggagaa
//
