LOCUS       sequence-401349-        6247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for the FYVE domain from ALFY for recombinant 
            expression in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6247)
  AUTHORS   Reinhart EF, Litt NA, Katzenell S, Pellegrini M, Yamamoto A, Ragusa 
            MJ
  TITLE     A highly conserved glutamic acid in ALFY inhibits membrane binding 
            to aid in aggregate clearance.
  JOURNAL   Traffic. 2021 Jan;22(1-2):23-37. doi: 10.1111/tra.12771. Epub 2020 
            Dec 1.
  PUBMED    33225481
REFERENCE   2  (bases 1 to 6247)
  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: "Traffic."; 
            date: "2021-01"; volume: "22(1-2):23-37. doi"; pages: " 
            10.1111/tra.12771. Epub 2020 Dec 1"
COMMENT     Sequence Label: ALFY-FYVE 1G
FEATURES             Location/Qualifiers
     source          1..6247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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)."
     RBS             3543..3565
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3584..3601
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3611..4264
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             4274..4294
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4595..4612
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4665..4683)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4679..4726
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4763..5218
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4850..4869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5060..5081
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5311..6126)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5311..6126)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6034..6053
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6118..6137
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat accctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3541 attttgttta actttaagaa ggagatatac catgggttct tctcaccatc accatcacca
     3601 tggttcttct atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac
     3661 tcgacttctt ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga
     3721 aggtgataaa tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta
     3781 ttatattgat ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga
     3841 caagcacaac atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg
     3901 agcggttttg gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac
     3961 tctcaaagtt gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt
     4021 atgtcataaa acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga
     4081 cgctcttgat gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt
     4141 ttgttttaaa aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa
     4201 gtatatagca tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc
     4261 aaaagggatc gaggaaaacc tgtacttcca atccaatgca ggccgttctg ctgctgatca
     4321 ctgggtgaag gatgaaggtg gtgacagctg ctcaggctgc tcggtgaggt tttcactcac
     4381 agaaagacga caccattgca ggaactgtgg tcagctcttc tgccagaagt gcagtcgctt
     4441 tcaatctgaa atcaaacgct tgaaaatctc atccccggtg cgtgtttgtc agaactgtta
     4501 ttataactta cagcatgaga gaggttaata acattggaag tggataacgg atccgaattc
     4561 gagcgccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
     4621 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     4681 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     4741 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4801 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     4861 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4921 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     4981 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5041 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5101 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5161 gagctgattt aacaaaaatt taacgcgaat tttaacaaac tagtaacgtt tacaatttca
     5221 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5281 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     5341 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     5401 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     5461 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     5521 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     5581 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     5641 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     5701 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     5761 atcaacaatg ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     5821 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     5881 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     5941 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     6001 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     6061 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     6121 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     6181 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6241 gatcttc
//
