LOCUS       sequence-274538-        6219 bp    DNA     circular SYN 15-APR-2025
DEFINITION  low-copy URA-selectable plasmid, 1xFLAG-MCP under TEF1 promoter, 
            ECM33 intron inserted into URA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6219)
  AUTHORS   Morgan JT, Fink GR, Bartel DP
  TITLE     Excised linear introns regulate growth in yeast.
  JOURNAL   Nature. 2019 Jan;565(7741):606-611. doi: 10.1038/s41586-018-0828-1. 
            Epub 2019 Jan 16.
  PUBMED    30651636
REFERENCE   2  (bases 1 to 6219)
  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/s41586-018-0828-1"; journalName: "Nature"; date: "2019-01";
            volume: "565"; issue: "7741"; pages: "606-611"
COMMENT     Sequence Label: pRS416-PTEF1-MCP-ECM33_intron
FEATURES             Location/Qualifiers
     source          1..6219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..22
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(241..345)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    382..885
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     926..944
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(986..1008)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1111..1130
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1154..1369
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     rep_origin      complement(2634..3089)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2771..2792)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2983..3002
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3215..3237
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3229..3246
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3230..3246
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3256..3275
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3256..3274
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3288..3686
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             3705..3728
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4122..4142
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4155..4173)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      4185..4374
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4399..4419)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4399..4417)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4438..4454)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4438..4454)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4451..4473)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4462..4478
                     /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(4486..4492,4493..4510,4511..4516))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4531..4552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4669..4686)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4840..5428)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4920..4939)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5599..6219,1..171,172..240))
                     /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"
ORIGIN
        1 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
       61 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
      121 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
      181 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
      241 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
      301 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      361 agtgccacct gggtcctttt catcacgtgc tataaaaata attataattt aaatttttta
      421 atataaatat ataaattaaa aatagaaagt aaaaaaagaa attaaagaaa aaatagtttt
      481 tgttttccga agatgtaaaa gactctaggg ggatcgccaa caaatactac cttttatctt
      541 gctcttcctg ctctcaggta ttaatgccga attgtttcat cttgtctgtg tagaagacca
      601 cacacgaaaa tcctgtgatt ttacatttta cttatcgtta atcgaatgta tatctattta
      661 atctgctttt cttgtctaat aaatatatat gtaaagtacg ctttttgttg aaatttttta
      721 aacctttgtt tatttttttt tcttcattcc gtaactcttc taccttcttt atttactttc
      781 taaaatccaa atacaaaaca taaaaataaa taaacacaga gtaaattccc aaattattcc
      841 atcattaaaa gatacgaggc gcgtgtaagt tacaggcaag cgatccgtct aagaaaccat
      901 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcgaattcg
      961 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag atctcggtca
     1021 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     1081 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ccgagagtgc
     1141 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
     1201 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
     1261 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
     1321 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
     1381 tacatataag gaacgtgctg ctactcatcc tagtcctgtt ggtatgtaca cattctcctt
     1441 ttttttcatc tttttttctt tatttgcctc tttcctcaca aatctcgagt agattcgtgg
     1501 tcctcttcat tcttttcttt tttctttgtc gattactggg ctttttttca taggtctcga
     1561 ttgacgcgga cggacaatgc gaaaaaaaaa aatttccaaa agaggaaacg ggtttcgaga
     1621 tctcgtgagg atggttttgg aatattcttc aatatttctg cctgtcatag gattagggcg
     1681 agtaatgaaa cagaataata catgtataaa tcgatcggga atactaacac tacttttctt
     1741 tatctaagca gctgccaagc tatttaatat catgcacgaa aagcaaacaa acttgtgtgc
     1801 ttcattggat gttcgtacca ccaaggaatt actggagtta gttgaagcat taggtcccaa
     1861 aatttgttta ctaaaaacac atgtggatat cttgactgat ttttccatgg agggcacagt
     1921 taagccgcta aaggcattat ccgccaagta caatttttta ctcttcgaag acagaaaatt
     1981 tgctgacatt ggtaatacag tcaaattgca gtactctgcg ggtgtataca gaatagcaga
     2041 atgggcagac attacgaatg cacacggtgt ggtgggccca ggtattgtta gcggtttgaa
     2101 gcaggcggca gaagaagtaa caaaggaacc tagaggcctt ttgatgttag cagaattgtc
     2161 atgcaagggc tccctatcta ctggagaata tactaagggt actgttgaca ttgcgaagag
     2221 cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa gagatgaagg
     2281 ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggtg atgcattggg
     2341 tcaacagtat agaaccgtgg atgatgtggt ctctacagga tctgacatta ttattgttgg
     2401 aagaggacta tttgcaaagg gaagggatgc taaggtagag ggtgaacgtt acagaaaagc
     2461 aggctgggaa gcatatttga gaagatgcgg ccagcaaaac taaaaaactg tattataagt
     2521 aaatgcatgt atactaaact cacaaattag agcttcaatt taattatatc agttattacc
     2581 ctatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt
     2641 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2701 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2761 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     2821 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     2881 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     2941 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3001 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3061 cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg
     3121 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     3181 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     3241 gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccat agcttcaaaa
     3301 tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac
     3361 ttcaaaacac ccaagcacag catactaaat ttcccctctt tcttcctcta gggtgtcgtt
     3421 aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg
     3481 tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaaattt tttttttgat
     3541 ttttttctct ttcgatgacc tcccattgat atttaagtta ataaacggtc ttcaatttct
     3601 caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt gctcattaga
     3661 aagaaagcat agcaatctaa tctaagtttt ctagaactag tatggactac aaggacgacg
     3721 atgacaagat ggcttctaac tttactcagt tcgttctcgt cgacaatggc ggaactggcg
     3781 acgtgactgt cgccccaagc aacttcgcta acggggtcgc tgaatggatc agctctaact
     3841 cgcgatcaca ggcttacaaa gtaacctgta gcgttcgtca gagctctgcg cagaatcgca
     3901 aatacaccat caaagtcgag gtgcctaaag tggcaaccca gactgttggt ggtgaagagc
     3961 ttcctgtagc cggatggaga tcttacttaa atatggaact aaccattcca attttcgcca
     4021 cgaattccga ctgcgagctt attgttaagg caatgcaagg tctcctaaaa gatggaaacc
     4081 cgattccctc ggccatcgcg gccaactccg gcatctaccc accaaaaaaa aaaagaaaag
     4141 tttagattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa
     4201 aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta
     4261 gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac
     4321 gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta
     4381 atttgccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     4441 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4501 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4561 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4621 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4681 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4741 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4801 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4861 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4921 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4981 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5041 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5101 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5161 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5221 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5281 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5341 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5401 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5461 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5521 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5581 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5641 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5701 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5761 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5821 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     5881 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     5941 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6001 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6061 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6121 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6181 tatgcggcga ccgagttgct cttgcccggc gtcaatacg
//
