LOCUS       sequence-234889-        6866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of GFP tagged TIS11B CPM2 (charge pattern 
            mutant 2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6866)
  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 6866)
  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-TIS11B CPM2
FEATURES             Location/Qualifiers
     source          1..6866
                     /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(2706..2723)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2712..2936
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2982..3410
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3069..3088)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3279..3300
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3419..3439)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3424..3753
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3604..3739
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3666..3685
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3766..4365
                     /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(3766..3785)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4262..4282
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4539..4660
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4576..4595)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4630..4649
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4709..4725)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4709..4725)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4722..4744)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4733..4749
                     /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(4757..4763,4764..4781,4782..4787))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4802..4823
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4940..4957)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5111..5699)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5191..5210)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5870..6661,6662..6730))
                     /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     6493..6512
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6731..6835)
                     /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 gaccaccacc ctcgtgtctg ccaccatctt cgacttgagc gaagttttat gcaagggtaa
     1681 caagatgctc aactatagtg ctcccagtgc agggggttgc ctgctggaca gaaaggcagt
     1741 gggcacccct gctggtgggg gcttccctgc ggcgcactca gtcaccctgc ccagctccaa
     1801 gttccaccag aaccagctcc tcagcagcct caagggtgag ccagcccccg ctctgagctc
     1861 ggcagacagc gccttcgcag acgcctcctt ctcggaaggg ggcgagcggc tgctgcccac
     1921 ccagaagcag cccggtggcg gccaggtcaa ctccagccgc tacaagacgg agctgtgccg
     1981 cccctttgag gaaaacggtg cctgtaagta cggggacaag tgccagttcg cacacggcat
     2041 ccacgagctc cgcagcctga cccgccaccc caagtacaag acggagctgt gccgcacctt
     2101 ccacaccatc ggcttttgcc cctacgggcc ccgctgccac ttcatccaca acgctgaaga
     2161 ggacgacgcc ctggccgggg ccgacgacct ctccgctgac gaccccgacc tccagcatag
     2221 ctttagcttt gctgggtttc ccagtgccgc tgccaccgcc gctgccaccg ggctgctgga
     2281 cagccccacg tccatcaccc caccccctat tctgagcgcc gatgacctcc tgggctcacc
     2341 taccctgccc gatggcacca ataacccttt tgccttctcc agccaggagc tggcaagcct
     2401 ctttgcccct agcatggggc tgcccggggg tggctccccg accaccttcc tcttccggcc
     2461 catgtccgag tcccctcaca tgtttgactc tccccccagc cctcaggatt ctctctcgga
     2521 ccaggagggc tacctgagca gctccagcag cagccacagt ggctcagact ccccgacctt
     2581 ggacaactca agacgcctgc ccatcttcag cagactttcc atctcagatg actaagaatt
     2641 ctgcagatat ccagcacagt ggcggccgct cgagtctaga gggcccgttt aaacccgctg
     2701 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2761 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2821 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2881 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2941 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     3001 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3061 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3121 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3181 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3241 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3301 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3361 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3421 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3481 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3541 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3601 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3661 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3721 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc caaccatgac cgagtacaag
     3781 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     3841 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     3901 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     3961 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     4021 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     4081 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     4141 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     4201 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     4261 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     4321 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgagcggg actctggggt
     4381 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4441 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4501 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4561 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4621 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4681 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4741 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4801 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4861 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4921 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4981 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5041 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5101 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5161 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5221 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5281 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5341 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5401 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5461 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5521 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5581 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5641 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5701 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5761 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5821 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5881 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5941 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6001 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6061 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6121 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6181 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6241 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6301 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6361 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6421 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6481 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6541 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6601 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6661 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6721 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6781 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6841 ttccccgaaa agtgccacct gacgtc
//
