LOCUS       sequence-456589-        7457 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7457)
  AUTHORS   Burd CG, Emr SD
  TITLE     Phosphatidylinositol(3)-phosphate signaling mediated by specific 
            binding to RING FYVE domains.
  JOURNAL   Mol Cell. 1998 Jul;2(1):157-62.
  PUBMED    9702203
REFERENCE   2  (bases 1 to 7457)
  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: "Mol Cell.";
            date: "1998-07"; volume: "2(1)"; pages: "157-62"
COMMENT     Sequence Label: pRS424GFP-FYVE(EEA1)
FEATURES             Location/Qualifiers
     source          1..7457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        191..472
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             473..1147
                     /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(1250..1705)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1387..1408)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1599..1618
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1831..1853
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1845..1862
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1846..1862
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1872..1891
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1872..1890
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2092..2280)
                     /codon_start=1
                     /product="FYVE domain of human early endosome antigen 1"
                     /label=FYVE(EEA1)
                     /note="binds PtdIns(3)P (Burd and Emr, 1998)"
                     /translation="WAEDNEVQNCMACGKGFSVTVRRHHCRQCGNIFCAECSAKNALTP
                     SSKKPVRVCDACFNDLQG"
     CDS             complement(2593..3306)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MGKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2633..2653)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     3248..3276
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(3802..3818)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3802..3818)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3855..3875)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3855..3873)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3894..3910)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3894..3910)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3907..3929)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3918..3934
                     /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(3942..3948,3949..3966,3967..3972))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3987..4008
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4125..4142)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4296..4884)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4376..4395)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5055..5846,5847..5915))
                     /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     5678..5697
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5916..6020)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6047..7389
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7010..7057)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     7430..7448
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatagatc aacgacatta ctatatatat aatataggaa gcatttaata gaacagcatc
      241 gtaatatatg tgtactttgc agttatgacg ccagatggca gtagtggaag atattcttta
      301 ttgaaaaata gcttgtcacc ttacgtacaa tcttgatccg gagcttttct ttttttgccg
      361 attaagaatt aattcggtcg aaaaaagaaa aggagagggc caagagggag ggcattggtg
      421 actattgagc acgtgagtat acgtgattaa gcacacaaag gcagcttgga gtatgtctgt
      481 tattaatttc acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga
      541 ggccgcagaa tgtgctctag attccgatgc tgacttgctg ggtattatat gtgtgcccaa
      601 tagaaagaga acaattgacc cggttattgc aaggaaaatt tcaagtcttg taaaagcata
      661 taaaaatagt tcaggcactc cgaaatactt ggttggcgtg tttcgtaatc aacctaagga
      721 ggatgttttg gctctggtca atgattacgg cattgatatc gtccaactgc atggagatga
      781 gtcgtggcaa gaataccaag agttcctcgg tttgccagtt attaaaagac tcgtatttcc
      841 aaaagactgc aacatactac tcagtgcagc ttcacagaaa cctcattcgt ttattccctt
      901 gtttgattca gaagcaggtg ggacaggtga acttttggat tggaactcga tttctgactg
      961 ggttggaagg caagagagcc ccgaaagctt acattttatg ttagctggtg gactgacgcc
     1021 agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt ggtgttgatg taagcggagg
     1081 tgtggagaca aatggtgtaa aagactctaa caaaatagca aatttcgtca aaaatgctaa
     1141 gaaataggtt attactgagt agtatttatt taagtattgt ttgtgcactt gccgatctat
     1201 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     1261 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     1321 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     1381 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     1441 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     1501 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     1561 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     1621 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     1681 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     1741 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     1801 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     1861 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg
     1921 tcgactctag aggatccccc atccataaca ttccaaaata tactaattcc tatttggtct
     1981 agtattgcaa ccagtgtcca aaccaaatag tagtccaaga cctctattaa gtacatttat
     2041 taaaaatcta atgttagtgt aatattactc tgaagttgtg ataacccatt atccttgcaa
     2101 gtcattgaaa catgcatcac agacacgaac aggcttcttg gaggaaggag ttaaggcatt
     2161 tttggctgaa cattcagcac agaagatatt tccacactgt cggcagtgat gccgtctcac
     2221 tgttactgaa aagcctttcc cacaggccat acagttttgt acttcattgt cttcggccca
     2281 ctttctattc aacgcttgtg tatgtttgat ctgaagtgat tggttttctc tgcccagctc
     2341 ctgcactgct gcagttgtat tatccagctt tctttgcaat tctaatactt tggtttgaag
     2401 cttttctatt tcaccttctc ctttaagaca tctttccagt agtgctctcc tttcatcttg
     2461 attattctga accgttgctt ctaatactgc aatttcaccc cgtaagtcat ccgtttgttt
     2521 ctcaagctca ctaactctcc gttgactaga ttgccacgaa ttcgaagctt gagctcgaga
     2581 tctgggtccg gacttgtata gttcatccat gccatgtgta atcccagcag ctgttacaaa
     2641 ctcaagaagg accatgtggt ctctcttttc gttgggatct ttcgaaaggg cagattgtgt
     2701 ggacaggtaa tggttgtctg gtaaaaggac agggccatcg ccaattggag tattttgttg
     2761 ataatggtct gctagttgaa cgcttccatc ttcaatgttg tgtctaattt tgaagttaac
     2821 tttgattcca ttcttttgtt tgtctgccat gatgtataca ttgtgtgagt tatagttgta
     2881 ttccaatttg tgtccaagaa tgtttccatc ttctttaaaa tcaatacctt ttaactcgat
     2941 tctattaaca agggtatcac cttcaaactt gacttcagca cgtgtcttgt agttcccgtc
     3001 atctttgaaa aatatagttc tttcctgtac ataaccttcg ggcatggcac tcttgaaaaa
     3061 gtcatgccgt ttcatatgat ctgggtatct tgaaaagcat tgaacaccat acgtaaaagt
     3121 agtgacaagt gttggccatg gaacaggtag ttttccagta gtgcaaataa atttaagggt
     3181 aagttttccg tatgttgcat caccttcacc ctctccactg acagaaaatt tgtgcccatt
     3241 aacatcacca tctaattcaa caagaattgg gacaactcca gtgaaaagtt cttctccttt
     3301 acccatagcg tatgtatact ttaagttgag tagaaaagaa acaatctcta gtgagtattc
     3361 tttctctctc tctctcttgt cttgttaaga ctaggaggga agacgaaaag aattatttcg
     3421 gagctttttt gctggcattc ttatctcgtg cttttattag tgggcccctt ctttgagacc
     3481 ccgcgggtga tatggcccct gtctcaatga tggataaagt tgttggaagc aattaccaag
     3541 acaatcacaa acgaaggttg ctaccgaagt gtactggatg accaacatcg ctatgattaa
     3601 ataagactat acataataga caagggtggc gcggctccat tcccagggag acacaaacac
     3661 gaggtaagtc tcaaaaaacc gaagtttcgc tctaaaactg cacgtaaagg tcgtggtatt
     3721 gactacaacc attgactgtt acattgtgta gaagaaaggt cctcacatac tgctctgtca
     3781 atatggcttc ttgtgcagaa ggatccacta gttctagagc ggccgccacc gcggtggagc
     3841 tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     3901 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3961 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4021 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4081 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4141 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4201 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4261 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4321 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4381 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4441 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4501 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4561 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4621 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4681 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4741 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4801 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4861 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4921 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4981 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5041 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5101 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5161 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5221 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5281 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5341 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5401 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5461 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5521 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5581 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5641 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5701 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5761 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5821 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5881 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5941 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6001 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaacg aagcatctgt
     6061 gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct
     6121 gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat
     6181 ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag
     6241 aatctgagct gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa
     6301 agaatctata cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac
     6361 aaagcatctt agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa
     6421 ctttttgcac tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc tattttctct
     6481 tccataaaaa aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt
     6541 gcattttttc aagataaagg catccccgat tatattctat accgatgtgg attgcgcata
     6601 ctttgtgaac agaaagtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg
     6661 tttcttctat tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt
     6721 tcgattcact ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag
     6781 ataaacataa aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat
     6841 gggtaggtta tatagggata tagcacagag atatatagca aagagatact tttgagcaat
     6901 gtttgtggaa gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg
     6961 ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat
     7021 actttctaga gaataggaac ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc
     7081 ttccgaaaat gcaacgcgag ctgcgcacat acagctcact gttcacgtcg cacctatatc
     7141 tgcgtgttgc ctgtatatat atatacatga gaagaacggc atagtgcgtg tttatgctta
     7201 aatgcgtact tatatgcgtc tatttatgta ggatgaaagg tagtctagta cctcctgtga
     7261 tattatccca ttccatgcgg ggtatcgtat gcttccttca gcactaccct ttagctgttc
     7321 tatatgctgc cactcctcaa ttggattagt ctcatccttc aatgctatca tttcctttga
     7381 tattggatca tactaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     7441 cacgaggccc tttcgtc
//
