LOCUS       sequence-274552-        6307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  low-copy URA-selectable plasmid, 1xFLAG-MCP under TEF1 promoter, 
            3P-ECM33 intron inserted into URA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6307)
  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 6307)
  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-3P-ECM33_intron
FEATURES             Location/Qualifiers
     source          1..6307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     163..181
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(223..245)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     348..367
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        391..606
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     misc_RNA        902..920
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        941..959
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      complement(1959..2414)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2096..2117)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2308..2327
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2540..2562
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2554..2571
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2555..2571
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2581..2600
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2581..2599
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2613..3011
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             3030..3053
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3447..3467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3480..3498)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      3510..3699
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3724..3744)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3724..3742)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3763..3779)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3763..3779)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3776..3798)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3787..3803
                     /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(3811..3817,3818..3835,3836..3841))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3856..3877
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3994..4011)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4165..4753)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4245..4264)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4924..5715,5716..5784))
                     /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     5547..5566
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5785..5889)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(5926..6307,1..122)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 cttctttatt tactttctaa aatccaaata caaaacataa aaataaataa acacagagta
       61 aattcccaaa ttattccatc attaaaagat acgaggcgcg tgtaagttac aggcaagcga
      121 tccgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
      181 ccctttcgtc gaattcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
      241 cccggagatc tcggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
      301 gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca
      361 gattgtaccg agagtgcacc ataccacctt ttcaattcat catttttttt ttattctttt
      421 ttttgatttc ggtttccttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa
      481 cgaaggaagg agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca
      541 tgaaattgcc cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga
      601 taaatcatgt cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttggt
      661 atgtacacat tctccttttt tttcatcttt ttttctttat ttgcctcttt cctcacaaat
      721 ctcgagtaga ttcgtggtcc tcttcattct tttctttttt ctttgtcgat tactgggctt
      781 tttttcatag gtctcgattg acgcggacgg acaatgcgaa aaaaaaaaat ttccaaaaga
      841 ggaaacgggt ttcgagatct cgtgaggatg gttttggaat attcgggatt tttgacgtcg
      901 cacatgagga tcacccatgt gcgacgtctt ttcacgagcg acatgaggat cacccatgtc
      961 gctcgtgttc ccttcaatat ttctgcctgt cataggatta gggcgagtaa tgaaacagaa
     1021 taatacatgt ataaatcgat cgggaatact aacactactt ttctttatct aagcagctgc
     1081 caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat tggatgttcg
     1141 taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt gtttactaaa
     1201 aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc cgctaaaggc
     1261 attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg acattggtaa
     1321 tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg cagacattac
     1381 gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg cggcagaaga
     1441 agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca agggctccct
     1501 atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca aagattttgt
     1561 tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg attggttgat
     1621 tatgacaccc ggtgtgggtt tagatgacaa gggtgatgca ttgggtcaac agtatagaac
     1681 cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag gactatttgc
     1741 aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct gggaagcata
     1801 tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg catgtatact
     1861 aaactcacaa attagagctt caatttaatt atatcagtta ttaccctatg cggtgtgaaa
     1921 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaatatttt
     1981 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     2041 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     2101 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     2161 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     2221 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     2281 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     2341 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     2401 gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     2461 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     2521 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg
     2581 taatacgact cactataggg cgaattgggt accatagctt caaaatgttt ctactccttt
     2641 tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa aacacccaag
     2701 cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta cccgtactaa
     2761 aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata
     2821 aaaattttta tcacgtttct ttttcttgaa aatttttttt ttgatttttt tctctttcga
     2881 tgacctccca ttgatattta agttaataaa cggtcttcaa tttctcaagt ttcagtttca
     2941 tttttcttgt tctattacaa ctttttttac ttcttgctca ttagaaagaa agcatagcaa
     3001 tctaatctaa gttttctaga actagtatgg actacaagga cgacgatgac aagatggctt
     3061 ctaactttac tcagttcgtt ctcgtcgaca atggcggaac tggcgacgtg actgtcgccc
     3121 caagcaactt cgctaacggg gtcgctgaat ggatcagctc taactcgcga tcacaggctt
     3181 acaaagtaac ctgtagcgtt cgtcagagct ctgcgcagaa tcgcaaatac accatcaaag
     3241 tcgaggtgcc taaagtggca acccagactg ttggtggtga agagcttcct gtagccggat
     3301 ggagatctta cttaaatatg gaactaacca ttccaatttt cgccacgaat tccgactgcg
     3361 agcttattgt taaggcaatg caaggtctcc taaaagatgg aaacccgatt ccctcggcca
     3421 tcgcggccaa ctccggcatc tacccaccaa aaaaaaaaag aaaagtttag attagttatg
     3481 tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga
     3541 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta
     3601 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata
     3661 ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg ccagcttttg
     3721 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     3781 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3841 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3901 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3961 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4021 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4081 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4141 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4201 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4261 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4321 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4381 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4441 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4501 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4561 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4621 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4681 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4741 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4801 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4861 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4921 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4981 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5041 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5101 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5161 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5221 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5281 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5341 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5401 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5461 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5521 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5581 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5641 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5701 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5761 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5821 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5881 ggttccgcgc acatttcccc gaaaagtgcc acctgggtcc ttttcatcac gtgctataaa
     5941 aataattata atttaaattt tttaatataa atatataaat taaaaataga aagtaaaaaa
     6001 agaaattaaa gaaaaaatag tttttgtttt ccgaagatgt aaaagactct agggggatcg
     6061 ccaacaaata ctacctttta tcttgctctt cctgctctca ggtattaatg ccgaattgtt
     6121 tcatcttgtc tgtgtagaag accacacacg aaaatcctgt gattttacat tttacttatc
     6181 gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc taataaatat atatgtaaag
     6241 tacgcttttt gttgaaattt tttaaacctt tgtttatttt tttttcttca ttccgtaact
     6301 cttctac
//
