LOCUS       sequence-274500-        5865 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  low-copy URA-selectable plasmid, 1xFLAG-MCP under TEF1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5865)
  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 5865)
  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-no_intron
FEATURES             Location/Qualifiers
     source          1..5865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..73
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     65..82
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     66..82
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     92..111
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        92..110
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        124..522
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             541..564
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(967..985)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      997..1186
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1211..1231)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1211..1229)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1250..1266)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1250..1266)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1263..1285)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1274..1290
                     /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(1298..1304,1305..1322,1323..1328))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1343..1364
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1481..1498)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1652..2240)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1732..1751)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2411..3202,3203..3271))
                     /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     3034..3053
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3272..3376)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    3413..3916
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3957..3975
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4017..4039)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4142..4161
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4185..4400
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             4401..5204
                     /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(5335..5790)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5472..5493)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5684..5703
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
       61 acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg
      121 taccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc tcggactccg
      181 cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaatttcc cctctttctt
      241 cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct
      301 cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga
      361 aaattttttt tttgattttt ttctctttcg atgacctccc attgatattt aagttaataa
      421 acggtcttca atttctcaag tttcagtttc atttttcttg ttctattaca acttttttta
      481 cttcttgctc attagaaaga aagcatagca atctaatcta agttttctag aactagtatg
      541 gactacaagg acgacgatga caagatggct tctaacttta ctcagttcgt tctcgtcgac
      601 aatggcggaa ctggcgacgt gactgtcgcc ccaagcaact tcgctaacgg ggtcgctgaa
      661 tggatcagct ctaactcgcg atcacaggct tacaaagtaa cctgtagcgt tcgtcagagc
      721 tctgcgcaga atcgcaaata caccatcaaa gtcgaggtgc ctaaagtggc aacccagact
      781 gttggtggtg aagagcttcc tgtagccgga tggagatctt acttaaatat ggaactaacc
      841 attccaattt tcgccacgaa ttccgactgc gagcttattg ttaaggcaat gcaaggtctc
      901 ctaaaagatg gaaacccgat tccctcggcc atcgcggcca actccggcat ctactagatt
      961 agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg
     1021 agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag
     1081 aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa
     1141 cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcca
     1201 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     1261 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     1321 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     1381 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     1441 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1501 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1561 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1621 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1681 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1741 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1801 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     1861 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     1921 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     1981 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2041 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2101 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2161 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2221 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2281 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2341 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2401 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2461 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     2521 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     2581 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     2641 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     2701 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     2761 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     2821 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     2881 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     2941 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3001 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3061 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3121 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3181 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3241 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3301 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3361 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgggtccttt tcatcacgtg
     3421 ctataaaaat aattataatt taaatttttt aatataaata tataaattaa aaatagaaag
     3481 taaaaaaaga aattaaagaa aaaatagttt ttgttttccg aagatgtaaa agactctagg
     3541 gggatcgcca acaaatacta ccttttatct tgctcttcct gctctcaggt attaatgccg
     3601 aattgtttca tcttgtctgt gtagaagacc acacacgaaa atcctgtgat tttacatttt
     3661 acttatcgtt aatcgaatgt atatctattt aatctgcttt tcttgtctaa taaatatata
     3721 tgtaaagtac gctttttgtt gaaatttttt aaacctttgt ttattttttt ttcttcattc
     3781 cgtaactctt ctaccttctt tatttacttt ctaaaatcca aatacaaaac ataaaaataa
     3841 ataaacacag agtaaattcc caaattattc catcattaaa agatacgagg cgcgtgtaag
     3901 ttacaggcaa gcgatccgtc taagaaacca ttattatcat gacattaacc tataaaaata
     3961 ggcgtatcac gaggcccttt cgtcgaattc gcgcgtttcg gtgatgacgg tgaaaacctc
     4021 tgacacatgc agctcccgga gatctcggtc acagcttgtc tgtaagcgga tgccgggagc
     4081 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     4141 gcggcatcag agcagattgt accgagagtg caccatacca ccttttcaat tcatcatttt
     4201 ttttttattc ttttttttga tttcggtttc cttgaaattt ttttgattcg gtaatctccg
     4261 aacagaagga agaacgaagg aaggagcaca gacttagatt ggtatatata cgcatatgta
     4321 gtgttgaaga aacatgaaat tgcccagtat tcttaaccca actgcacaga acaaaaacct
     4381 gcaggaaacg aagataaatc atgtcgaaag ctacatataa ggaacgtgct gctactcatc
     4441 ctagtcctgt tgctgccaag ctatttaata tcatgcacga aaagcaaaca aacttgtgtg
     4501 cttcattgga tgttcgtacc accaaggaat tactggagtt agttgaagca ttaggtccca
     4561 aaatttgttt actaaaaaca catgtggata tcttgactga tttttccatg gagggcacag
     4621 ttaagccgct aaaggcatta tccgccaagt acaatttttt actcttcgaa gacagaaaat
     4681 ttgctgacat tggtaataca gtcaaattgc agtactctgc gggtgtatac agaatagcag
     4741 aatgggcaga cattacgaat gcacacggtg tggtgggccc aggtattgtt agcggtttga
     4801 agcaggcggc agaagaagta acaaaggaac ctagaggcct tttgatgtta gcagaattgt
     4861 catgcaaggg ctccctatct actggagaat atactaaggg tactgttgac attgcgaaga
     4921 gcgacaaaga ttttgttatc ggctttattg ctcaaagaga catgggtgga agagatgaag
     4981 gttacgattg gttgattatg acacccggtg tgggtttaga tgacaagggt gatgcattgg
     5041 gtcaacagta tagaaccgtg gatgatgtgg tctctacagg atctgacatt attattgttg
     5101 gaagaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag
     5161 caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact gtattataag
     5221 taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac
     5281 cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg
     5341 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     5401 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     5461 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     5521 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     5581 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     5641 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     5701 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     5761 ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg
     5821 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggc
//
