LOCUS       sequence-274543-        6305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  low-copy URA-selectable plasmid, 1xFLAG-MCP under TEF1 promoter, 
            5P-ECM33 intron inserted into URA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6305)
  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 6305)
  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: 
            "10.1038/s41586-018-0828-1"; journalName: "Nature"; date: "2019-01";
            volume: "565"; issue: "7741"; pages: "606-611"
COMMENT     Sequence Label: pRS416-PTEF1-MCP-5P-ECM33_intron
FEATURES             Location/Qualifiers
     source          1..6305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(15..37)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     140..159
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        183..398
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     misc_RNA        527..545
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        564..582
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      complement(1749..2204)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1886..1907)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2098..2117
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2330..2352
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2344..2361
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2345..2361
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2371..2390
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2371..2389
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2403..2801
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             2820..2843
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3237..3257
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3270..3288)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      3300..3489
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3514..3534)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3514..3532)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3553..3569)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3553..3569)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3566..3588)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3577..3593
                     /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(3601..3607,3608..3625,3626..3631))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3646..3667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3784..3801)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3955..4543)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4035..4054)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4714..5505,5506..5574))
                     /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     5337..5356
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5575..5679)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5716..6219
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6260..6278
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gatgacggtg aaaacctctg acacatgcag ctcccggaga tctcggtcac agcttgtctg
       61 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
      121 cggggctggc ttaactatgc ggcatcagag cagattgtac cgagagtgca ccataccacc
      181 ttttcaattc atcatttttt ttttattctt ttttttgatt tcggtttcct tgaaattttt
      241 ttgattcggt aatctccgaa cagaaggaag aacgaaggaa ggagcacaga cttagattgg
      301 tatatatacg catatgtagt gttgaagaaa catgaaattg cccagtattc ttaacccaac
      361 tgcacagaac aaaaacctgc aggaaacgaa gataaatcat gtcgaaagct acatataagg
      421 aacgtgctgc tactcatcct agtcctgttg gtatgtacac attctccttt tttttcatct
      481 ttttttcttt atttgcctct ttcctcacag ggatttttga cgtcgcacat gaggatcacc
      541 catgtcgacg tcttttcacg gcgacatgag gatcacccat gtcgctcgtg ttcccaatct
      601 cgagtagatt cgtggtcctc ttcattcttt tcttttttct ttgtcgatta ctgggctttt
      661 tttcataggt ctcgattgac gcggacggac aatgcgaaaa aaaaaaattt ccaaaagagg
      721 aaacgggttt cgagatctcg tgaggatggt tttggaatat tcttcaatat ttctgcctgt
      781 cataggatta gggcgagtaa tgaaacagaa taatacatgt ataaatcgat cgggaatact
      841 aacactactt ttctttatct aagcagctgc caagctattt aatatcatgc acgaaaagca
      901 aacaaacttg tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga
      961 agcattaggt cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc
     1021 catggagggc acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt
     1081 cgaagacaga aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt
     1141 atacagaata gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat
     1201 tgttagcggt ttgaagcagg cggcagaaga agtaacaaag gaacctagag gccttttgat
     1261 gttagcagaa ttgtcatgca agggctccct atctactgga gaatatacta agggtactgt
     1321 tgacattgcg aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg
     1381 tggaagagat gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa
     1441 gggtgatgca ttgggtcaac agtatagaac cgtggatgat gtggtctcta caggatctga
     1501 cattattatt gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga
     1561 acgttacaga aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa
     1621 aactgtatta taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt
     1681 atatcagtta ttaccctatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     1741 gcatcaggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     1801 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     1861 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     1921 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     1981 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     2041 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2101 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2161 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg
     2221 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     2281 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     2341 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt
     2401 accatagctt caaaatgttt ctactccttt tttactcttc cagattttct cggactccgc
     2461 gcatcgccgt accacttcaa aacacccaag cacagcatac taaatttccc ctctttcttc
     2521 ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc
     2581 gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa
     2641 aatttttttt ttgatttttt tctctttcga tgacctccca ttgatattta agttaataaa
     2701 cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac
     2761 ttcttgctca ttagaaagaa agcatagcaa tctaatctaa gttttctaga actagtatgg
     2821 actacaagga cgacgatgac aagatggctt ctaactttac tcagttcgtt ctcgtcgaca
     2881 atggcggaac tggcgacgtg actgtcgccc caagcaactt cgctaacggg gtcgctgaat
     2941 ggatcagctc taactcgcga tcacaggctt acaaagtaac ctgtagcgtt cgtcagagct
     3001 ctgcgcagaa tcgcaaatac accatcaaag tcgaggtgcc taaagtggca acccagactg
     3061 ttggtggtga agagcttcct gtagccggat ggagatctta cttaaatatg gaactaacca
     3121 ttccaatttt cgccacgaat tccgactgcg agcttattgt taaggcaatg caaggtctcc
     3181 taaaagatgg aaacccgatt ccctcggcca tcgcggccaa ctccggcatc tacccaccaa
     3241 aaaaaaaaag aaaagtttag attagttatg tcacgcttac attcacgccc tccccccaca
     3301 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt
     3361 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
     3421 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
     3481 cgctcgaagg ctttaatttg ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
     3541 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3601 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3661 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3721 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3781 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3841 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3901 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3961 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4021 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4081 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4141 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4201 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4261 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4321 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4381 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4441 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4501 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4561 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4621 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4681 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4741 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4801 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4861 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4921 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4981 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5041 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5101 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5161 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5221 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5281 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5341 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5401 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5461 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5521 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5581 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5641 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5701 acctgggtcc ttttcatcac gtgctataaa aataattata atttaaattt tttaatataa
     5761 atatataaat taaaaataga aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt
     5821 ccgaagatgt aaaagactct agggggatcg ccaacaaata ctacctttta tcttgctctt
     5881 cctgctctca ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag accacacacg
     5941 aaaatcctgt gattttacat tttacttatc gttaatcgaa tgtatatcta tttaatctgc
     6001 ttttcttgtc taataaatat atatgtaaag tacgcttttt gttgaaattt tttaaacctt
     6061 tgtttatttt tttttcttca ttccgtaact cttctacctt ctttatttac tttctaaaat
     6121 ccaaatacaa aacataaaaa taaataaaca cagagtaaat tcccaaatta ttccatcatt
     6181 aaaagatacg aggcgcgtgt aagttacagg caagcgatcc gtctaagaaa ccattattat
     6241 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcgaa ttcgcgcgtt
     6301 tcggt
//
