LOCUS       sequence-464955-        7397 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of full-length hnRNPA1 D262V with C-terminal 
            FLAG tag co-expressing with eGFP via IRES sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7397)
  AUTHORS   Das T, Zaidi F, Farag M, Ruff KM, Messing J, Taylor JP, Pappu RV, 
            Mittag T
  TITLE     Metastable condensates suppress conversion to amyloid fibrils.
  JOURNAL   bioRxiv [Preprint]. 2024 Mar 3:2024.02.28.582569. doi: 
            10.1101/2024.02.28.582569.
  PUBMED    38464104
REFERENCE   2  (bases 1 to 7397)
  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: "bioRxiv 
            [Preprint]. 2024 Mar 3:2024.02.28.582569. doi: 
            10.1101/2024.02.28.582569."
COMMENT     Sequence Label: hnRNPA1_D262V-FLAG-IRES-eGFP
FEATURES             Location/Qualifiers
     source          1..7397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        135..182
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             201..575
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    705..826
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(742..761)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     796..815
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(875..891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(875..891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(888..910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    899..915
                     /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(923..929,930..947,948..953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    968..989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1106..1123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1277..1865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1357..1376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2036..2827,2828..2896))
                     /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     2659..2678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2897..3001)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3076..3095)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3267..3646
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3647..3850
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3801..3821
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    3852..3870
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3873..3891
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3901..3925
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             4845..4856
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             5017..5040
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    5062..5611
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5206..5223)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5433..5452
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(5614..5616,5617..5619,5620..6330)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5614..5616,5617..5619,5620..6330)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5659..5680)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5920..5939)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6267..6288
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6352..6381
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6397..6414
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6437..6454)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6443..6667
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6713..7141
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6800..6819)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7010..7031
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7150..7170)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(7155..7397,1..87)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(7335..7397,1..73)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     join(7397,1..19)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
       61 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
      121 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
      181 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
      241 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
      301 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
      361 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
      421 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
      481 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
      541 actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
      601 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
      661 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
      721 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
      781 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
      841 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      901 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
      961 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1021 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1081 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1141 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1201 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1261 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1321 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1381 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1441 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1501 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1561 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1621 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1681 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1741 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1801 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1861 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1921 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1981 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2041 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2101 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2161 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2221 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2281 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2341 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2401 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2461 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2521 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2581 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2641 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     2701 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2761 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2821 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2881 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2941 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3001 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     3061 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     3121 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     3181 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     3241 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     3301 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     3361 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     3421 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     3481 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     3541 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     3601 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     3661 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     3721 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     3781 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     3841 aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag atcgtcgacg
     3901 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca
     3961 tagaagacac cgggaccgat ccagcctccg gactctagcg tttaaactta agcttggtac
     4021 cgagctcgga tccactagtc cagtgtggtg gaattcatgt ctaagtcaga gtctcctaaa
     4081 gagcccgaac agctgaggaa gctcttcatt ggagggttga gctttgaaac aactgatgag
     4141 agcctgagga gccattttga gcaatgggga acgctcacgg actgtgtggt aatgagagat
     4201 ccaaacacca agcgctccag gggctttggg tttgtcacat atgccactgt ggaggaggtg
     4261 gatgcagcta tgaatgcaag gccacacaag gtggatggaa gagttgtgga accaaagaga
     4321 gctgtctcca gagaagattc tcaaagacca ggtgcccact taactgtgaa aaagatattt
     4381 gttggtggca ttaaagaaga cactgaagaa catcacctaa gagattattt tgaacagtat
     4441 ggaaaaattg aagtgattga aatcatgact gaccgaggca gtggcaagaa aaggggcttt
     4501 gcctttgtaa cctttgacga ccatgactcc gtggataaga ttgtcattca gaaataccat
     4561 actgtgaatg gccacaactg tgaagttaga aaagccctgt caaagcaaga gatggctagt
     4621 gcttcatcca gccaaagagg tcgaagtggt tctggaaact ttggtggtgg tcgtggaggt
     4681 ggtttcggtg ggaatgacaa cttcggtcgt ggaggaaact tcagtggtcg tggtggcttt
     4741 ggtggcagcc gtggtggtgg tggatatggt ggcagtgggg atggctataa tggatttggt
     4801 aatgatggaa gcaattttgg aggtggtgga agctacaatg tttttgggaa ttacaacaat
     4861 cagtcttcaa attttggacc catgaaggga ggaaattttg gaggcagaag ctctggcccc
     4921 tatggcggtg gaggccaata ctttgcaaaa ccacgaaacc aaggtggcta tggcggttcc
     4981 agcagcagca gtagctatgg cagtggcaga agatttgact acaaggacga cgatgacaag
     5041 taagcggccg ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     5101 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     5161 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     5221 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     5281 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     5341 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     5401 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     5461 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     5521 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg
     5581 acgtggtttt cctttgaaaa acacgataat accatggtga gcaagggcga ggagctgttc
     5641 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     5701 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     5761 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     5821 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     5881 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     5941 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     6001 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     6061 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     6121 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     6181 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     6241 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     6301 atcactctcg gcatggacga gctgtacaag ctcgagtcta gagggccctt cgaacaaaaa
     6361 ctcatctcag aagaggatct gaatatgcat accggtcatc atcaccatca ccattgagtt
     6421 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     6481 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     6541 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     6601 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     6661 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     6721 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     6781 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     6841 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     6901 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     6961 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     7021 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     7081 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     7141 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     7201 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     7261 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     7321 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     7381 gctgactaat ttttttt
//
