LOCUS       sequence-477258-        8627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cytosolic expression of mEmerald.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8627)
  AUTHORS   Goldman, C., Kareva, T., Sarrafha, L., Schuldt, B.R., Sahasrabudhe, 
            A., Ahfeldt, T.,, Blanchard, J.W
  TITLE     Genetically encoded and modular subcellular organelle probes reveal 
            dysfunction in lysosomes and mitochondria driven by PRKN knockout
  JOURNAL   iScience 28 (2025)
REFERENCE   2  (bases 1 to 8627)
  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: "iScience 28
            (2025)"
COMMENT     Sequence Label: pFUW mEmerald-P2A-PuroR
FEATURES             Location/Qualifiers
     source          1..8627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(48..67)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        239..618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        620..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     773..793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             836..1016
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1063..1188
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1681..1914
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2099..2143
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2441..2558
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2623..3834
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3671..3690
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      3865..3874
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3871..3873,3874..3876,3877..4587)
                     /codon_start=1
                     /product="brightly fluorescent monomeric variant of GFP"
                     /label=mEmerald
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3916..3937)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4177..4196)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4524..4545
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4612..4668
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4669..5268
                     /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(4669..4688)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5165..5185
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5294..5882
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5347..5367)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5765..5776)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5885..5901)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5886..5902)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6407..6587
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6613..6630)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6619..6730
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(6876..7464)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6956..6975)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7635..8426,8427..8495))
                     /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     8258..8277
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8496..8600)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
       61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      241 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      421 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
      841 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      901 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      961 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     1021 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     1081 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     1141 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     1201 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     1261 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     1321 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1381 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1441 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1501 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1561 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1621 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1681 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1741 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1801 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1861 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1921 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1981 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     2041 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     2101 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     2161 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     2221 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     2281 atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga
     2341 agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg
     2401 tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     2461 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     2521 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     2581 gagatccagt ttggttaatt aacccgtgtc ggctccagat ctggcctccg cgccgggttt
     2641 tggcgcctcc cgcgggcgcc cccctcctca cggcgagcgc tgccacgtca gacgaagggc
     2701 gcagcgagcg tcctgatcct tccgcccgga cgctcaggac agcggcccgc tgctcataag
     2761 actcggcctt agaaccccag tatcagcaga aggacatttt aggacgggac ttgggtgact
     2821 ctagggcact ggttttcttt ccagagagcg gaacaggcga ggaaaagtag tcccttctcg
     2881 gcgattctgc ggagggatct ccgtggggcg gtgaacgccg atgattatat aaggacgcgc
     2941 cgggtgtggc acagctagtt ccgtcgcagc cgggatttgg gtcgcggttc ttgtttgtgg
     3001 atcgctgtga tcgtcacttg gtgagtagcg ggctgctggg ctggccgggg ctttcgtggc
     3061 cgccgggccg ctcggtggga cggaagcgtg tggagagacc gccaagggct gtagtctggg
     3121 tccgcgagca aggttgccct gaactggggg ttggggggag cgcagcaaaa tggcggctgt
     3181 tcccgagtct tgaatggaag acgcttgtga ggcgggctgt gaggtcgttg aaacaaggtg
     3241 gggggcatgg tgggcggcaa gaacccaagg tcttgaggcc ttcgctaatg cgggaaagct
     3301 cttattcggg tgagatgggc tggggcacca tctggggacc ctgacgtgaa gtttgtcact
     3361 gactggagaa ctcggtttgt cgtctgttgc gggggcggca gttatggcgg tgccgttggg
     3421 cagtgcaccc gtacctttgg gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg
     3481 ttggcttata atgcagggtg gggccacctg ccggtaggtg tgcggtaggc ttttctccgt
     3541 cgcaggacgc agggttcggg cctagggtag gctctcctga atcgacaggc gccggacctc
     3601 tggtgagggg agggataagt gaggcgtcag tttctttggt cggttttatg tacctatctt
     3661 cttaagtagc tgaagctccg gttttgaact atgcgctcgg ggttggcgag tgtgttttgt
     3721 gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt
     3781 tagactagta aattgtccgc taaattctgg ccgtttttgg cttttttgtt agacgaagct
     3841 tgggcccggg aattaattat taaggccacc atggtgagca agggcgagga gctgttcacc
     3901 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     3961 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     4021 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttgaccta cggcgtgcag
     4081 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     4141 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     4201 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     4261 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaag
     4321 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg tgaacttcaa gacccgccac
     4381 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     4441 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa gctgagcaaa
     4501 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     4561 actctcggca tggacgagct gtacaaggat ctgcagtcta gaggatctgg tgccacgaat
     4621 ttctcactac tcaagcaggc cggtgatgtc gaggaaaacc ctggtcctat gaccgagtac
     4681 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     4741 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     4801 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     4861 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     4921 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     4981 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     5041 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     5101 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     5161 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     5221 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg aattcgatat
     5281 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     5341 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     5401 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     5461 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     5521 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     5581 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     5641 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     5701 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     5761 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     5821 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     5881 gcatcgatac cgtcgacctc gagacctaga aaaacatgga gcaatcacaa gtagcaatac
     5941 agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt
     6001 tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
     6061 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     6121 tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc agaactacac
     6181 accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
     6241 tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt
     6301 gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg
     6361 cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt
     6421 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     6481 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     6541 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa
     6601 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     6661 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     6721 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     6781 acagcaaggg ggaggattgg gaagacaata gcaggcatgt gagcaaaagg ccagcaaaag
     6841 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6901 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6961 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     7021 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     7081 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     7141 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     7201 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     7261 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     7321 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7381 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7441 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     7501 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     7561 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     7621 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7681 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7741 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7801 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7861 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7921 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7981 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     8041 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     8101 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     8161 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     8221 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     8281 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     8341 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     8401 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     8461 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     8521 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     8581 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctga
//
