LOCUS       sequence-73674-e        6751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  overespression of Flp step-arrest mutant in yeast, TRP1 selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6751)
  AUTHORS   Tsalik EL, Gartenberg MR
  TITLE     Curing Saccharomyces cerevisiae of the 2 micron plasmid by targeted 
            DNA damage.
  JOURNAL   Yeast. 1998 Jun 30;14(9):847-52.
  PUBMED    9818722
REFERENCE   2  (bases 1 to 6751)
  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"; journalName: "Yeast."; 
            date: "1998-06-30"; volume: "14(9)"; pages: "847-52"
COMMENT     Sequence Label: pBIS-GALkFLP(Trp1)
FEATURES             Location/Qualifiers
     source          1..6751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..1272
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=FLP
                     /note="FLP is a site-specific recombinase from 
                     Saccharomyces cerevisiae. Recombination occurs at FRT 
                     sequences."
                     /translation="MPQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
                     THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
                     IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
                     SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
                     KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
                     SYNKALKKNAPYSIFAIKNGPKSLIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
                     TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
                     YPAWNGIISQEVLDYLSSYINRRI"
     primer_bind     complement(1500..1520)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1500..1518)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1539..1555)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1539..1555)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1552..1574)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1563..1579
                     /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(1587..1593,1594..1611,1612..1617))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(1770..1787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1941..2529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2021..2040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2700..3491,3492..3560))
                     /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     3323..3342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3561..3665)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    3702..4205
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     4247..4265
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4303..4325)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4425..4444
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4461..4741
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             4742..5416
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(5515..5970)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5652..5673)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5864..5883
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6100..6122
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6114..6131
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6115..6131
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6141..6160
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6141..6159
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6184..6200
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6185..6201
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6414..6744
                     /gene="S. cerevisiae GAL10"
                     /label=GAL10 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    6414..6531
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
ORIGIN
        1 atgccacaat ttggtatatt atgtaaaaca ccacctaagg tgcttgttcg tcagtttgtg
       61 gaaaggtttg aaagaccttc aggtgagaaa atagcattat gtgctgctga actaacctat
      121 ttatgttgga tgattacaca taacggaaca gcaatcaaga gagccacatt catgagctat
      181 aatactatca taagcaattc gctgagtttc gatattgtca ataaatcact ccagtttaaa
      241 tacaagacgc aaaaagcaac aattctggaa gcctcattaa agaaattgat tcctgcttgg
      301 gaatttacaa ttattcctta ctatggacaa aaacatcaat ctgatatcac tgatattgta
      361 agtagtttgc aattacagtt cgaatcatcg gaagaagcag ataagggaaa tagccacagt
      421 aaaaaaatgc ttaaagcact tctaagtgag ggtgaaagca tctgggagat cactgagaaa
      481 atactaaatt cgtttgagta tacttcgaga tttacaaaaa caaaaacttt ataccaattc
      541 ctcttcctag ctactttcat caattgtgga agattcagcg atattaagaa cgttgatccg
      601 aaatcattta aattagtcca aaataagtat ctgggagtaa taatccagtg tttagtgaca
      661 gagacaaaga caagcgttag taggcacata tacttcttta gcgcaagggg taggatcgat
      721 ccacttgtat atttggatga atttttgagg aattctgaac cagtcctaaa acgagtaaat
      781 aggaccggca attcttcaag caataaacag gaataccaat tattaaaaga taacttagtc
      841 agatcgtaca ataaagcttt gaagaaaaat gcgccttatt caatctttgc tataaaaaat
      901 ggcccaaaat ctctcattgg aagacatttg atgacctcat ttctttcaat gaagggccta
      961 acggagttga ctaatgttgt gggaaattgg agcgataagc gtgcttctgc cgtggccagg
     1021 acaacgtata ctcatcagat aacagcaata cctgatcact acttcgcact agtttctcgg
     1081 tactatgcat atgatccaat atcaaaggaa atgatagcat tgaaggatga gactaatcca
     1141 attgaggagt ggcagcatat agaacagcta aagggtagtg ctgaaggaag catacgatac
     1201 cccgcatgga atgggataat atcacaggag gtactagact acctttcatc ctacataaat
     1261 agacgcatat aagtacgcat ttaagcataa acacgcacta tgccgttctt ctcatgtata
     1321 tatatataca ggcaacacgc agatataggt gcgacgtgaa cagtgagctg tatgtgcgca
     1381 gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg ctttgaagtt cctattccga
     1441 agttcctatt ctctagttct agagcggccg ccaccgcggt ggagctccag cttttgttcc
     1501 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     1561 aattgttatc cgctcacaat tccacacaac ataggagccg gaagcataaa gtgtaaagcc
     1621 tggggtgcct aatgagtgag gtaactcaca ttaattgcgt tgcgctcact gcccgctttc
     1681 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1741 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     1801 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     1861 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1921 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1981 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2041 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2101 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2161 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2221 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2281 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2341 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     2401 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2461 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2521 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     2581 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     2641 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2701 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2761 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2821 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     2881 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2941 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3001 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3061 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3121 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3181 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3241 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3301 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3361 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3421 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3481 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3541 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     3601 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3661 ccgcgcacat ttccccgaaa agtgccacct gggtcctttt catcacgtgc tataaaaata
     3721 attataattt aaatttttta atataaatat ataaattaaa aatagaaagt aaaaaaagaa
     3781 attaaagaaa aaatagtttt tgttttccga agatgtaaaa gactctaggg ggatcgccaa
     3841 caaatactac cttttatctt gctcttcctg ctctcaggta ttaatgccga attgtttcat
     3901 cttgtctgtg tagaagacca cacacgaaaa tcctgtgatt ttacatttta cttatcgtta
     3961 atcgaatgta tatctattta atctgctttt cttgtctaat aaatatatat gtaaagtacg
     4021 ctttttgttg aaatttttta aacctttgtt tatttttttt tcttcattcc gtaactcttc
     4081 taccttcttt atttactttc taaaatccaa atacaaaaca taaaaataaa taaacacaga
     4141 gtaaattccc aaattattcc atcattaaaa gatacgaggc gcgtgtaagt tacaggcaag
     4201 cgatccgtcc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     4261 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     4321 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     4381 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     4441 tgtactgaga gtgcaccata aacgacatta ctatatatat aatataggaa gcatttaata
     4501 gacagcatcg taatatatgt gtactttgca gttatgacgc cagatggcag tagtggaaga
     4561 tattctttat tgaaaaatag cttgtcacct tacgtacaat cttgatccgg agcttttctt
     4621 tttttgccga ttaagaatta attcggtcga aaaaagaaaa ggagagggcc aagagggagg
     4681 gcattggtga ctattgagca cgtgagtata cgtgattaag cacacaaagg cagcttggag
     4741 tatgtctgtt attaatttca caggtagttc tggtccattg gtgaaagttt gcggcttgca
     4801 gagcacagag gccgcagaat gtgctctaga ttccgatgct gacttgctgg gtattatatg
     4861 tgtgcccaat agaaagagaa caattgaccc ggttattgca aggaaaattt caagtcttgt
     4921 aaaagcatat aaaaatagtt caggcactcc gaaatacttg gttggcgtgt ttcgtaatca
     4981 acctaaggag gatgttttgg ctctggtcaa tgattacggc attgatatcg tccaactgca
     5041 tggagatgag tcgtggcaag aataccaaga gttcctcggt ttgccagtta ttaaaagact
     5101 cgtatttcca aaagactgca acatactact cagtgcagct tcacagaaac ctcattcgtt
     5161 tattcccttg tttgattcag aagcaggtgg gacaggtgaa cttttggatt ggaactcgat
     5221 ttctgactgg gttggaaggc aagagagccc cgaaagctta cattttatgt tagctggtgg
     5281 actgacgcca gaaaatgttg gtgatgcgct tagattaaat ggcgttattg gtgttgatgt
     5341 aagcggaggt gtggagacaa atggtgtaaa agactctaac aaaatagcaa atttcgtcaa
     5401 aaatgctaag aaataggtta ttactgagta gtatttattt aagtattgtt tgtgcacttg
     5461 cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg
     5521 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     5581 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     5641 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     5701 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     5761 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     5821 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     5881 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     5941 ccgcgcttaa tgcgccgcta cagggcgcgt cgcgccattc gccattcagg ctgcgcaact
     6001 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     6061 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     6121 cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt accgggcccc
     6181 ccctcgaggt cgacggtatc gataagcttg atatcgaatt cctgcagccc gggggatcca
     6241 aaaatcatcg cttcgctgat taattacccc agaaataagg ctaaaaaact aatcgcatta
     6301 tcatcctatg gttgttaatt tgattcgttc atttgaaggt ttgtggggcc aggttactgc
     6361 caatttttcc tcttcataac cataaaagct agtattgtag aatctttatt gttcggagca
     6421 gtgcggcgcg aggcacatct gcgtttcagg aacgcgaccg gtgaagacga ggacgcacgg
     6481 aggagagtct tccttcggag ggctgtcacc cgctcggcgg cttctaatcc gtacttcaat
     6541 atagcaatga gcagttaagc gtattactga aagttccaaa gagaaggttt ttttaggcta
     6601 agataatggg gctctttaca tttccacaac atataagtaa gattagatat ggatatgtat
     6661 atggatatgt atatggtggt aatgccatgt aatatgatta ttaaacttct ttgcgtccat
     6721 ccaaaaaaaa agtaagaatt tttgggtcga c
//
