LOCUS       sequence-255833-        6101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template for genomic C-terminal Cub-R-URA3 tag, URA3 complements
            the uracil auxotrophy of S. cerevisiae ura mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6101)
  AUTHORS   Dunkler A, Rosler R, Kestler HA, Moreno-Andres D, Johnsson N
  TITLE     SPLIFF: A Single-Cell Method to Map Protein-Protein Interactions in 
            Time and Space.
  JOURNAL   Methods Mol Biol. 2015;1346:151-68. doi: 
            10.1007/978-1-4939-2987-0_11.
  PUBMED    26542721
REFERENCE   2  (bases 1 to 6101)
  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"; journalName: "Methods Mol
            Biol."; date: "2015"; volume: "1346:151-68. doi"; pages: " 
            10.1007/978-1-4939-2987-0_11"
COMMENT     Sequence Label: pCCG 306
FEATURES             Location/Qualifiers
     source          1..6101
                     /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"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1963..1982
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1963..1981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2014..2030
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2014..2030
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             2087..2794
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2232..2250)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2500..2519
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3068..3094
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3107..3820
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MGKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3137..3165)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3760..3780
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        complement(3841..3859)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3880..3896)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3880..3896)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3893..3915)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3904..3920
                     /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(3928..3934,3935..3952,3953..3958))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3973..3994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4111..4128)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4282..4870)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4362..4381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5041..5832,5833..5901))
                     /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     5664..5683
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5902..6006)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6074..6092
                     /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 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     1981 ggcgaattgg agctccaccg cggtggcggc cgctctagaa ctagtggatc ccccgggctg
     2041 caggaattcg atatcaagct tatcgatacc gtcgaccggc gcgccaatgg tgagcaaggg
     2101 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     2161 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     2221 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     2281 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     2341 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     2401 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     2461 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     2521 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     2581 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     2641 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     2701 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     2761 ccactccacc ggcggcatgg acgagctgta caagggctcg accggtatcc ctccagatca
     2821 acaaagattg atctttgccg gtaagcagct agaagacggt agaacgctgt ctgattacaa
     2881 cattcagaag gagtccacct tacatcttgt gctaaggcta agaggtggta ggcacggatc
     2941 cggagcttgg ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     3001 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     3061 ccgtggctac ccatacgacg tcccagatta cgccgctggc gcgccaatgg gtaaaggaga
     3121 agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca
     3181 caaattttct gtcagtggag agggtgaagg cgatgcaaca tacggaaaac ttacccttaa
     3241 atttatttgc actactggaa aactacctgt tccatggcca acacttgtca ctactttcac
     3301 ctatggtgtt caatgctttt caagataccc agatcatatg aaacggcatg actttttcaa
     3361 gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa
     3421 ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt
     3481 aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta
     3541 taactcacac aatgtataca tcatggcaga caaacaaaag aatggaatca aagccaactt
     3601 caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa
     3661 tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc
     3721 tgccctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac
     3781 agctgctggg attacacatg gcatggatga actatacaaa taaggtacca gcttttgttc
     3841 cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     3901 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     3961 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4021 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4081 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4141 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4201 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4261 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4321 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4381 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4441 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4501 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4561 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4621 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4681 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4741 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4801 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4861 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4921 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4981 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5041 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5101 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5161 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5221 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5281 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5341 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5401 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5461 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5521 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5581 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5641 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5701 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5761 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5821 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5881 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5941 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6001 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     6061 attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
