LOCUS       sequence-466518-        5797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Restriction digest from pUC18-NFLAG-Fyv6 and ligation into pRS414.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5797)
  TITLE     Hoskins lab plasmids 2025
REFERENCE   2  (bases 1 to 5797)
  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"
COMMENT     Sequence Label: pRS414-NFLAG-Fyv6
FEATURES             Location/Qualifiers
     source          1..5797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(210..229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     329..351
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(389..407)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    448..951
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        988..1092
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1093..1161,1162..1953)
                     /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     complement(1311..1330)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2124..2712
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2613..2632
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2866..2883
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3000..3021
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3036..3041,3042..3059,3060..3066)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3074..3090
                     /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)."
     primer_bind     3079..3101
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3098..3114
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3098..3114
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3133..3153
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3135..3153
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(3970..3993)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4287..4306)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4288..4306)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4316..4333)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4316..4332)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4325..4347)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4473..4928
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4560..4579)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4770..4791
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5031..5705)
                     /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"
     promoter        complement(join(5706..5797,1..189))
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
ORIGIN
        1 cgaccgaatt aattcttaat cggcaaaaaa agaaaagctc cggatcaaga ttgtacgtaa
       61 ggtgacaagc tatttttcaa taaagaatat cttccactac tgccatctgg cgtcataact
      121 gcaaagtaca catatattac gatgctgttc tattaaatgc ttcctatatt atatatatag
      181 taatgtcgtg atctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
      241 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
      301 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
      361 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
      421 gtcatgataa taatggtttc ttagacggat cgcttgcctg taacttacac gcgcctcgta
      481 tcttttaatg atggaataat ttgggaattt actctgtgtt tatttatttt tatgttttgt
      541 atttggattt tagaaagtaa ataaagaagg tagaagagtt acggaatgaa gaaaaaaaaa
      601 taaacaaagg tttaaaaaat ttcaacaaaa agcgtacttt acatatatat ttattagaca
      661 agaaaagcag attaaataga tatacattcg attaacgata agtaaaatgt aaaatcacag
      721 gattttcgtg tgtggtcttc tacacagaca agatgaaaca attcggcatt aatacctgag
      781 agcaggaaga gcaagataaa aggtagtatt tgttggcgat ccccctagag tcttttacat
      841 cttcggaaaa caaaaactat tttttcttta atttcttttt ttactttcta tttttaattt
      901 atatatttat attaaaaaat ttaaattata attattttta tagcacgtga tgaaaaggac
      961 ccaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1021 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     1081 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     1141 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     1201 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     1261 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     1321 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     1381 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     1441 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     1501 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     1561 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     1621 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     1681 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     1741 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     1801 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     1861 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     1921 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     1981 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     2041 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2101 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2161 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2221 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     2281 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2341 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2401 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2461 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2521 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2581 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2641 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2701 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2761 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2821 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     2881 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     2941 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     3001 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     3061 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     3121 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct ccaccgcggt
     3181 ggcggccgcg tggaacgagc aatcaatgtg atcaatttgc catttcattc cacgaaatgg
     3241 tttccattct aagcatgaat gctttgacgt tacgggtgtt ggttatttcg gttcttttta
     3301 ttgcttccgt gggcacgtac gagaagggaa gatgaataga aaaagagttt ccgagttttg
     3361 tatgtccaat gtttacataa tttatataac ttgaagttta atcttggcat aaagctctag
     3421 ttgtgacgaa gtgtgtaggc catcacatca gttctccagt tttctaccca atttttttat
     3481 ggataccttg atttttggtt tagggctgga tgtctttacg ccctttattt gtgttttact
     3541 gcccaaacta gtgccggtct ctaaatattt ttctgtatta gtagtagaat cgttctccat
     3601 tagtaatgct ttttttttct tttcaaaatt gattgccttg acgtctaagt agttattcag
     3661 ttctttttcc ttttttagca actcattctt gctcttctgg taaaattcca ggtcctcttt
     3721 actcaaccta gtaaaacttt cacggtctct caccaaaccg ttatattgtt tttgtttact
     3781 aatagcatta gacctaagct gatctcgcag ggacttcctg gttttccttt tatattccgc
     3841 ttcatatttt ttttgctcca cttgctctcg tatcctctgg gcctctacat taccaactcc
     3901 ttctgacacg aatttcagtg gttttttctc tcttgcacta ttagcattat tatcactggt
     3961 agatgacatc ttgtcgtcat cgtctttgta gtccatgatg atataacaga gcccacagtc
     4021 ttttgttgga accgctttct ctaggcttcg atggacttac atctaattgc acattctcgt
     4081 gcgtttaaac gtcatttttc cctcagattg gcagtgcatc aaaagtaatt cctagggacg
     4141 acaaaaaggc ggaaaaggtt tcagaaaaat aagcggactt agctcagttg ggagagcgcc
     4201 agactgaaga aaaacttcgg tcaagtcatc tggaggtcct gtgttcgatc cagtcgacct
     4261 cgaggggggg cccggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     4321 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4381 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4441 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc
     4501 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4561 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4621 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4681 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4741 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4801 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4861 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     4921 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4981 gatcggcaag tgcacaaaca atacttaaat aaatactact cagtaataac ctatttctta
     5041 gcatttttga cgaaatttgc tattttgtta gagtctttta caccatttgt ctccacacct
     5101 ccgcttacat caacaccaat aacgccattt aatctaagcg catcaccaac attttctggc
     5161 gtcagtccac cagctaacat aaaatgtaag ctttcggggc tctcttgcct tccaacccag
     5221 tcagaaatcg agttccaatc caaaagttca cctgtcccac ctgcttctga atcaaacaag
     5281 ggaataaacg aatgaggttt ctgtgaagct gcactgagta gtatgttgca gtcttttgga
     5341 aatacgagtc ttttaataac tggcaaaccg aggaactctt ggtattcttg ccacgactca
     5401 tctccatgca gttggacgat atcaatgccg taatcattga ccagagccaa aacatcctcc
     5461 ttaggttgat tacgaaacac gccaaccaag tatttcggag tgcctgaact atttttatat
     5521 gcttttacaa gacttgaaat tttccttgca ataaccgggt caattgttct ctttctattg
     5581 ggcacacata taatacccag caagtcagca tcggaatcta gagcacattc tgcggcctct
     5641 gtgctctgca agccgcaaac tttcaccaat ggaccagaac tacctgtgaa attaataaca
     5701 gacatactcc aagctgcctt tgtgtgctta atcacgtata ctcacgtgct caatagtcac
     5761 caatgccctc cctcttggcc ctctcctttt ctttttt
//
