LOCUS       sequence-274545-        6187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  low-copy URA-selectable plasmid, 1xFLAG-MCP under TEF1 promoter, 
            5M-ECM33 intron inserted into URA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6187)
  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 6187)
  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-5M-ECM33_intron
FEATURES             Location/Qualifiers
     source          1..6187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     490..508
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(550..572)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     675..694
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        718..933
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     misc_RNA        1062..1080
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1101..1119
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      complement(2166..2621)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2303..2324)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2515..2534
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2747..2769
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2761..2778
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2762..2778
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2788..2807
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2788..2806
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2820..3218
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             3237..3260
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3654..3674
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3687..3705)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      3717..3906
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3931..3951)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3931..3949)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3970..3986)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3970..3986)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3983..4005)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3994..4010
                     /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(4018..4024,4025..4042,4043..4048))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4063..4084
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4201..4218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4372..4960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4452..4471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5131..5922,5923..5991))
                     /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     5754..5773
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5992..6096)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(6133..6187,1..449)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 taaaaataga aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt ccgaagatgt
       61 aaaagactct agggggatcg ccaacaaata ctacctttta tcttgctctt cctgctctca
      121 ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag accacacacg aaaatcctgt
      181 gattttacat tttacttatc gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc
      241 taataaatat atatgtaaag tacgcttttt gttgaaattt tttaaacctt tgtttatttt
      301 tttttcttca ttccgtaact cttctacctt ctttatttac tttctaaaat ccaaatacaa
      361 aacataaaaa taaataaaca cagagtaaat tcccaaatta ttccatcatt aaaagatacg
      421 aggcgcgtgt aagttacagg caagcgatcc gtctaagaaa ccattattat catgacatta
      481 acctataaaa ataggcgtat cacgaggccc tttcgtcgaa ttcgcgcgtt tcggtgatga
      541 cggtgaaaac ctctgacaca tgcagctccc ggagatctcg gtcacagctt gtctgtaagc
      601 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
      661 ctggcttaac tatgcggcat cagagcagat tgtaccgaga gtgcaccata ccaccttttc
      721 aattcatcat ttttttttta ttcttttttt tgatttcggt ttccttgaaa tttttttgat
      781 tcggtaatct ccgaacagaa ggaagaacga aggaaggagc acagacttag attggtatat
      841 atacgcatat gtagtgttga agaaacatga aattgcccag tattcttaac ccaactgcac
      901 agaacaaaaa cctgcaggaa acgaagataa atcatgtcga aagctacata taaggaacgt
      961 gctgctactc atcctagtcc tgttggtatg tacacattct cctttttttt catctttttt
     1021 tctttatttg cctctttcct cacagggatt tttgacgtcg cacatgagga tcacccatgt
     1081 gcgacgtctt ttcacgagcg acatgaggat cacccatgtc gctcgtgttc ccagaggaaa
     1141 cgggtttcga gatctcgtga ggatggtttt ggaatattct tcaatatttc tgcctgtcat
     1201 aggattaggg cgagtaatga aacagaataa tacatgtata aatcgatcgg gaatactaac
     1261 actacttttc tttatctaag cagctgccaa gctatttaat atcatgcacg aaaagcaaac
     1321 aaacttgtgt gcttcattgg atgttcgtac caccaaggaa ttactggagt tagttgaagc
     1381 attaggtccc aaaatttgtt tactaaaaac acatgtggat atcttgactg atttttccat
     1441 ggagggcaca gttaagccgc taaaggcatt atccgccaag tacaattttt tactcttcga
     1501 agacagaaaa tttgctgaca ttggtaatac agtcaaattg cagtactctg cgggtgtata
     1561 cagaatagca gaatgggcag acattacgaa tgcacacggt gtggtgggcc caggtattgt
     1621 tagcggtttg aagcaggcgg cagaagaagt aacaaaggaa cctagaggcc ttttgatgtt
     1681 agcagaattg tcatgcaagg gctccctatc tactggagaa tatactaagg gtactgttga
     1741 cattgcgaag agcgacaaag attttgttat cggctttatt gctcaaagag acatgggtgg
     1801 aagagatgaa ggttacgatt ggttgattat gacacccggt gtgggtttag atgacaaggg
     1861 tgatgcattg ggtcaacagt atagaaccgt ggatgatgtg gtctctacag gatctgacat
     1921 tattattgtt ggaagaggac tatttgcaaa gggaagggat gctaaggtag agggtgaacg
     1981 ttacagaaaa gcaggctggg aagcatattt gagaagatgc ggccagcaaa actaaaaaac
     2041 tgtattataa gtaaatgcat gtatactaaa ctcacaaatt agagcttcaa tttaattata
     2101 tcagttatta ccctatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     2161 tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2221 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2281 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2341 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     2401 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     2461 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     2521 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     2581 caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg
     2641 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     2701 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     2761 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc
     2821 atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca
     2881 tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc
     2941 tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt
     3001 tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat
     3061 tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg
     3121 tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc
     3181 ttgctcatta gaaagaaagc atagcaatct aatctaagtt ttctagaact agtatggact
     3241 acaaggacga cgatgacaag atggcttcta actttactca gttcgttctc gtcgacaatg
     3301 gcggaactgg cgacgtgact gtcgccccaa gcaacttcgc taacggggtc gctgaatgga
     3361 tcagctctaa ctcgcgatca caggcttaca aagtaacctg tagcgttcgt cagagctctg
     3421 cgcagaatcg caaatacacc atcaaagtcg aggtgcctaa agtggcaacc cagactgttg
     3481 gtggtgaaga gcttcctgta gccggatgga gatcttactt aaatatggaa ctaaccattc
     3541 caattttcgc cacgaattcc gactgcgagc ttattgttaa ggcaatgcaa ggtctcctaa
     3601 aagatggaaa cccgattccc tcggccatcg cggccaactc cggcatctac ccaccaaaaa
     3661 aaaaaagaaa agtttagatt agttatgtca cgcttacatt cacgccctcc ccccacatcc
     3721 gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt
     3781 atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac
     3841 agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc
     3901 tcgaaggctt taatttgcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     3961 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4021 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4081 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4141 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4201 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4261 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4321 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4381 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4441 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4501 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4561 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4621 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4681 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4741 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4801 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4861 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4921 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4981 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5041 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5101 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5161 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5221 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5281 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5341 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5401 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5461 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5521 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5581 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5641 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5701 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5761 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5821 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5881 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5941 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6001 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6061 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6121 tgggtccttt tcatcacgtg ctataaaaat aattataatt taaatttttt aatataaata
     6181 tataaat
//
