LOCUS       sequence-314503-        6989 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  overexpression of Flp recombinase step-arrest mutant in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6989)
  TITLE     Yeast viruses
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6989)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGAL-flp(H305L)-NAT
FEATURES             Location/Qualifiers
     source          1..6989
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..331
                     /gene="S. cerevisiae GAL10"
                     /label=GAL10 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    1..118
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     CDS             339..1610
                     /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="MPQFDILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
                     THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
                     IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
                     SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
                     KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
                     SYNKALKKNAPYSIFAIKNGPKSLIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
                     TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
                     YPAWNGIISQEVLDYLSSYINRRI"
     primer_bind     complement(1838..1858)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1838..1856)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1877..1893)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1877..1893)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1890..1912)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1901..1917
                     /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(1925..1931,1932..1949,1950..1955))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1970..1991
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2108..2125)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2279..2867)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2359..2378)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3038..3829,3830..3898))
                     /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     3661..3680
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3899..4003)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    4040..4543
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     4584..4602
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4640..4662)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4762..4781
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            complement(4860..5979)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(4860..5057)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(5063..5635)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(5636..5979)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      complement(6096..6551)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6233..6254)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6445..6464
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6677..6699
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6691..6708
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6692..6708
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6718..6737
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6718..6736
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6761..6777
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6762..6778
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cggagcagtg cggcgcgagg cacatctgcg tttcaggaac gcgaccggtg aagacgagga
       61 cgcacggagg agagtcttcc ttcggagggc tgtcacccgc tcggcggctt ctaatccgta
      121 cttcaatata gcaatgagca gttaagcgta ttactgaaag ttccaaagag aaggtttttt
      181 taggctaaga taatggggct ctttacattt ccacaacata taagtaagat tagatatgga
      241 tatgtatatg gatatgtata tggtggtaat gccatgtaat atgattatta aacttctttg
      301 cgtccatcca aaaaaaaagt aagaattttt gagtcgacat gccacaattt gatatattat
      361 gtaaaacacc acctaaggtg cttgttcgtc agtttgtgga aaggtttgaa agaccttcag
      421 gtgagaaaat agcattatgt gctgctgaac taacctattt atgttggatg attacacata
      481 acggaacagc aatcaagaga gccacattca tgagctataa tactatcata agcaattcgc
      541 tgagtttcga tattgtcaat aaatcactcc agtttaaata caagacgcaa aaagcaacaa
      601 ttctggaagc ctcattaaag aaattgattc ctgcttggga atttacaatt attccttact
      661 atggacaaaa acatcaatct gatatcactg atattgtaag tagtttgcaa ttacagttcg
      721 aatcatcgga agaagcagat aagggaaata gccacagtaa aaaaatgctt aaagcacttc
      781 taagtgaggg tgaaagcatc tgggagatca ctgagaaaat actaaattcg tttgagtata
      841 cttcgagatt tacaaaaaca aaaactttat accaattcct cttcctagct actttcatca
      901 attgtggaag attcagcgat attaagaacg ttgatccgaa atcatttaaa ttagtccaaa
      961 ataagtatct gggagtaata atccagtgtt tagtgacaga gacaaagaca agcgttagta
     1021 ggcacatata cttctttagc gcaaggggta ggatcgatcc acttgtatat ttggatgaat
     1081 ttttgaggaa ttctgaacca gtcctaaaac gagtaaatag gaccggcaat tcttcaagca
     1141 ataaacagga ataccaatta ttaaaagata acttagtcag atcgtacaat aaagctttga
     1201 agaaaaatgc gccttattca atctttgcta taaaaaatgg cccaaaatct ctcattggaa
     1261 gacatttgat gacctcattt ctttcaatga agggcctaac ggagttgact aatgttgtgg
     1321 gaaattggag cgataagcgt gcttctgccg tggccaggac aacgtatact catcagataa
     1381 cagcaatacc tgatcactac ttcgcactag tttctcggta ctatgcatat gatccaatat
     1441 caaaggaaat gatagcattg aaggatgaga ctaatccaat tgaggagtgg cagcatatag
     1501 aacagctaaa gggtagtgct gaaggaagca tacgataccc cgcatggaat gggataatat
     1561 cacaggaggt actagactac ctttcatcct acataaatag acgcatataa gtacgcattt
     1621 aagcataaac acgcactatg ccgttcttct catgtatata tatatacagg caacacgcag
     1681 atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc tcgcgttgca ttttcggaag
     1741 cgctcgtttt cggaaacgct ttgaagttcc tattccgaag ttcctattct ctagttctag
     1801 agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc
     1861 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1921 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1981 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2041 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2101 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2161 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2221 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2281 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2341 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2401 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2461 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2521 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2581 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2641 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2701 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2761 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2821 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2881 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2941 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3001 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3061 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3121 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3181 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3241 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3301 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3361 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3421 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3481 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3541 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3601 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3661 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3721 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3781 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3841 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3901 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3961 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4021 tgccacctgg gtccttttca tcacgtgcta taaaaataat tataatttaa attttttaat
     4081 ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg
     4141 ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct tttatcttgc
     4201 tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta gaagaccaca
     4261 cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata tctatttaat
     4321 ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa attttttaaa
     4381 cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat ttactttcta
     4441 aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa attattccat
     4501 cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtctaa gaaaccatta
     4561 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     4621 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     4681 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     4741 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccataatc
     4801 atcgatgaat tcgagctcgt tttcgacact ggatggcggc gttagtatcg aatcgacagc
     4861 agtatagcga ccagcattca catacgattg acgcatgata ttactttctg cgcacttaac
     4921 ttcgcatctg ggcagatgat gtcgaggcga aaaaaaatat aaatcacgct aacatttgat
     4981 taaaatagaa caactacaat ataaaaaaac tatacaaatg acaagttctt gaaaacaaga
     5041 atctttttat tgtcagtact gattaggggc agggcatgct catgtagagc gcctgctcgc
     5101 cgtccgaggc ggtgccgtcg tacagggcgg tgtccaggcc gcagagggtg aaccccatcc
     5161 gccggtacgc gtggatcgcc ggtgcgttga cgttggtgac ctccagccag aggtgcccgg
     5221 cgccccgctc gcgggcgaac tccgtcgcga gccccatcaa cgcgcgcccg accccgtgcc
     5281 cccggtgctc cggggcgacc tcgatgtcct cgacggtcag ccggcggttc cagccggagt
     5341 acgagacgac cacgaagccc gccaggtcgc cgtcgtcccc gtacgcgacg aacgtccggg
     5401 agtccgggtc gccgtcctcc ccgtcgtccg attcgtcgtc cgattcgtcg tcggggaaca
     5461 ccttggtcag gggcgggtcc accggcacct cccgcagggt gaagccgtcc ccggtggcgg
     5521 tgacgcggaa gacggtgtcg gtggtgaagg acccatccag tgcctcgatg gcctcggcgt
     5581 cccccgggac actggtgcgg taccggtaag ccgtgtcgtc aagagtggta cccatggttg
     5641 tttatgttcg gatgtgatgt gagaactgta tcctagcaag attttaaaag gaagtatatg
     5701 aaagaagaac ctcagtggca aatcctaacc ttttatattt ctctacaggg gcgcggcgtg
     5761 gggacaattc aacgcgtctg tgaggggagc gtttccctgc tcgcaggtct gcagcgagga
     5821 gccgtaattt ttgcttcgcg ccgtgcggcc atcaaaatgt atggatgcaa atgattatac
     5881 atggggatgt atgggctaaa tgtacgggcg acagtcacat catgcccctg agctgcgcac
     5941 gtcaagactg tcaaggaggg tattctgggc ctccatgtcg ctggccgggt gacccggcgg
     6001 ggacgaggca agctaaacag atctggcgcg ccttaattaa ccctatgcgg tgtgaaatac
     6061 cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt
     6121 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     6181 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     6241 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     6301 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     6361 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     6421 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     6481 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     6541 acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     6601 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     6661 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa
     6721 tacgactcac tatagggcga attgggtacc gggccccccc tcgaggtcga cggtatcgat
     6781 aagcttgata tcgaattcct gcagcccggg ggatccaaaa tcatcgcttc gctgattaat
     6841 taccccagaa ataaggctaa aaaactaatc gcattatcat cctatggttg ttaatttgat
     6901 tcgttcattt gaaggtttgt ggggccaggt tactgccaat ttttcctctt cataaccata
     6961 aaagctagta ttgtagaatc tttattgtt
//
