LOCUS       sequence-234829-        7751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of GFP tagged HSPA8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7751)
  AUTHORS   Ma W, Mayr C
  TITLE     A Membraneless Organelle Associated with the Endoplasmic Reticulum 
            Enables 3'UTR-Mediated Protein-Protein Interactions.
  JOURNAL   Cell. 2018 Oct 30. pii: S0092-8674(18)31315-1. doi: 
            10.1016/j.cell.2018.10.007.
  PUBMED    30449617
REFERENCE   2  (bases 1 to 7751)
  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: "Cell. 2018 
            Oct 30. pii: S0092-8674(18)31315-1. doi: 
            10.1016/j.cell.2018.10.007."
COMMENT     Sequence Label: GFP-HSPA8
FEATURES             Location/Qualifiers
     source          1..7751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(905..907,908..910,911..1621)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(950..971)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1211..1230)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1558..1579
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3591..3608)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3597..3821
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3867..4295
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3954..3973)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4164..4185
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4304..4324)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4309..4638
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4489..4624
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4551..4570
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4651..5250
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4651..4670)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5147..5167
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5424..5545
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5461..5480)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5515..5534
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5594..5610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5594..5610)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5607..5629)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5618..5634
                     /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(5642..5648,5649..5666,5667..5672))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5687..5708
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5825..5842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5996..6584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6076..6095)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6755..7546,7547..7615))
                     /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     7378..7397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7616..7720)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 aaccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      961 ggacggcgac gtaaacggcc acaagttcag cgtgtctggc gagggcgagg gcgatgccac
     1021 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1081 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1141 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1201 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1261 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1321 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1381 gaacggcatc aaggcgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1441 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1501 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1561 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1621 gtccaaggga cctgcagttg gtattgatct tggcaccacc tactcttgtg tgggtgtttt
     1681 ccagcacgga aaagtcgaga taattgccaa tgatcaggga aaccgaacca ctccaagcta
     1741 tgtcgccttt acggacactg aacggttgat cggtgatgcc gcaaagaatc aagttgcaat
     1801 gaaccccacc aacacagttt ttgatgccaa acgtctgatt ggacgcagat ttgatgatgc
     1861 tgttgtccag tctgatatga aacattggcc ctttatggtg gtgaatgatg ctggcaggcc
     1921 caaggtccaa gtagaataca agggagagac caaaagcttc tatccagagg aggtgtcttc
     1981 tatggttctg acaaagatga aggaaattgc agaagcctac cttgggaaga ctgttaccaa
     2041 tgctgtggtc acagtgccag cttactttaa tgactctcag cgtcaggcta ccaaagatgc
     2101 tggaactatt gctggtctca atgtacttag aattattaat gagccaactg ctgctgctat
     2161 tgcttacggc ttagacaaaa aggttggagc agaaagaaac gtgctcatct ttgacctggg
     2221 aggtggcact tttgatgtgt caatcctcac tattgaggat ggaatctttg aggtcaagtc
     2281 tacagctgga gacacccact tgggtggaga agattttgac aaccgaatgg tcaaccattt
     2341 tattgctgag tttaagcgca agcataagaa ggacatcagt gagaacaaga gagctgtaag
     2401 acgcctccgt actgcttgtg aacgtgctaa gcgtaccctc tcttccagca cccaggccag
     2461 tattgagatc gattctctct atgaaggaat cgacttctat acctccatta cccgtgcccg
     2521 atttgaagaa ctgaatgctg acctgttccg tggcaccctg gacccagtag agaaagccct
     2581 tcgagatgcc aaactagaca agtcacagat tcatgatatt gtcctggttg gtggttctac
     2641 tcgtatcccc aagattcaga agcttctcca agacttcttc aatggaaaag aactgaataa
     2701 gagcatcaac cctgatgaag ctgttgctta tggtgcagct gtccaggcag ccatcttgtc
     2761 tggagacaag tctgagaatg ttcaagattt gctgctcttg gatgtcactc ctctttccct
     2821 tggtattgaa actgctggtg gagtcatgac tgtcctcatc aagcgtaata ccaccattcc
     2881 taccaagcag acacagacct tcactaccta ttctgacaac cagcctggtg tgcttattca
     2941 ggtttatgaa ggcgagcgtg ccatgacaaa ggataacaac ctgcttggca agtttgaact
     3001 cacaggcata cctcctgcac cccgaggtgt tcctcagatt gaagtcactt ttgacattga
     3061 tgccaatggt atactcaatg tctctgctgt ggacaagagt acgggaaaag agaacaagat
     3121 tactatcact aatgacaagg gccgtttgag caaggaagac attgaacgta tggtccagga
     3181 agctgagaag tacaaagctg aagatgagaa gcagagggac aaggtgtcat ccaagaattc
     3241 acttgagtcc tatgccttca acatgaaagc aactgttgaa gatgagaaac ttcaaggcaa
     3301 gattaacgat gaggacaaac agaagattct ggacaagtgt aatgaaatta tcaactggct
     3361 tgataagaat cagactgctg agaaggaaga atttgaacat caacagaaag agctggagaa
     3421 agtttgcaac cccatcatca ccaagctgta ccagagtgca ggaggcatgc caggaggaat
     3481 gcctggggga tttcctggtg gtggagctcc tccctctggt ggtgcttcct cagggcccac
     3541 cattgaagag gttgattaat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     3601 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3661 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3721 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3781 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3841 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3901 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3961 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4021 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4081 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4141 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4201 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4261 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     4321 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4381 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4441 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4501 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4561 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4621 ggcctaggct tttgcaaaaa gctcccaacc atgaccgagt acaagcccac ggtgcgcctc
     4681 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     4741 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     4801 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     4861 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4921 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     4981 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     5041 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     5101 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     5161 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     5221 tggtgcatga cccgcaagcc cggtgcctga gcgggactct ggggttcgaa atgaccgacc
     5281 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     5341 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5401 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     5461 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5521 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     5581 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5641 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5701 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5761 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5821 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5881 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5941 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6001 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6061 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6121 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6181 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6241 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6301 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6361 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6421 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6481 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6541 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6601 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6661 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6721 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6781 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6841 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6901 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6961 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7021 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7081 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7141 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7201 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7261 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7321 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7381 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7441 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7501 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7561 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7621 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7681 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7741 cacctgacgt c
//
