LOCUS       sequence-382363-        5927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GST- and mCherry-tagged HRS FYVE in e Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5927)
  AUTHORS   Jensen LE, Rao S, Schuschnig M, Cada AK, Martens S, Hummer G, Hurley
            JH
  TITLE     Membrane curvature sensing and stabilization by the autophagic LC3 
            lipidation machinery.
  JOURNAL   Sci Adv. 2022 Dec 14;8(50):eadd1436. doi: 10.1126/sciadv.add1436. 
            Epub 2022 Dec 14.
  PUBMED    36516251
REFERENCE   2  (bases 1 to 5927)
  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: "Sci Adv."; 
            date: "2022-12-14"; volume: "8(50):eadd1436. doi"; pages: " 
            10.1126/sciadv.add1436. Epub 2022 Dec 14"
COMMENT     Sequence Label: GST-mCh-FYVE
FEATURES             Location/Qualifiers
     source          1..5927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(131..136,137..154,155..161)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    169..185
                     /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     193..209
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     193..209
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             205..732
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(225..242)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(225..241)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(234..256)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(834..839,840..855,856..862)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    870..886
                     /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)."
     CDS             909..1562
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1520..1542
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1575..1595
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1617..2324
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1762..1780)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2030..2049
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2628..2650)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2795..2813)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2881..2985
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2986..3054,3055..3846)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3204..3223)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4017..4605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4506..4525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4759..4776
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4849..4926
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4927..5927,1..82)
                     /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     complement(4946..4965)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
       61 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
      121 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      181 aacaatttca cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta
      241 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
      301 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
      361 cgcagcctga atggcgaatg gcgctttgcc tggtttccgg caccagaagc ggtgccggaa
      421 agctggctgg agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc aaactggcag
      481 atgcacggtt acgatgcgcc catctacacc aacgtgacct atcccattac ggtcaatccg
      541 ccgtttgttc ccacggagaa tccgacgggt tgttactcgc tcacatttaa tgttgatgaa
      601 agctggctac aggaaggcca gacgcgaatt atttttgatg gcgttggaat tagcttatcg
      661 actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg
      721 tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa
      781 tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa
      841 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
      901 cagtattcat gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc
      961 gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag
     1021 gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt
     1081 atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca
     1141 agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag
     1201 cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc
     1261 tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat
     1321 gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg
     1381 ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt
     1441 gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt
     1501 atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa
     1561 aaatcgatac gaccgaaaac ctgtattttc agggcgccat gggatccgga attcaaatgg
     1621 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     1681 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1741 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
     1801 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     1861 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     1921 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     1981 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     2041 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     2101 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     2161 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     2221 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     2281 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagggtgct cagatgtttg
     2341 ctgccgagag agccccagac tgggtggacg ctgaggaatg ccaccgctgc agggtgcagt
     2401 tcggggtgat gacccgtaag caccactgcc gggcgtgtgg gcagatattc tgtggaaagt
     2461 gttcttccaa gtactccacc atccccaagt ttggcatcga gaaggaggtg cgcgtgtgtg
     2521 agccctgcta cgagcagctg aacaggaaag cggagggata atctcgagcg gccgcatcgt
     2581 gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     2641 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     2701 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     2761 gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     2821 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     2881 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2941 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3001 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3061 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3121 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3181 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     3241 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3301 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3361 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3421 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3481 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     3541 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3601 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3661 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3721 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3781 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3841 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3901 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3961 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4021 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4081 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4141 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4201 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4261 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4321 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4381 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4441 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4501 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4561 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4621 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4681 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4741 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4801 tattttctcc ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa
     4861 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
     4921 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
     4981 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     5041 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     5101 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     5161 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     5221 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     5281 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     5341 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     5401 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     5461 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     5521 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     5581 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     5641 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     5701 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     5761 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     5821 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     5881 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatac
//
