LOCUS       sequence-493222-        8594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a secretome mRNA reporter: MS2 mRNA encoding 45aa of the 
            Type II membrane protein, sialyltransferase (a Golgi enzyme), fused 
            with EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8594)
  AUTHORS   Choi H, Liao YC, Yoon YJ, Grimm J, Wang N, Lavis LD, Singer RH, 
            Lippincott-Schwartz J
  TITLE     Secretome translation shaped by lysosomes and lunapark-marked ER 
            junctions.
  JOURNAL   Nature. 2025 Nov 5. doi: 10.1038/s41586-025-09718-0.
  PUBMED    41193816
REFERENCE   2  (bases 1 to 8594)
  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: "Nature. 
            2025 Nov 5. doi: 10.1038/s41586-025-09718-0."
COMMENT     Sequence Label: pUBC-SiT-EGFP-MS2
FEATURES             Location/Qualifiers
     source          1..8594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    148..169
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(184..189,190..207,208..214)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    222..238
                     /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     227..249
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     246..262
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     246..262
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     312..331
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             523..1156
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    1203..1328
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1825..2058
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2243..2287
                     /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    2580..2697
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2740..3138
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     regulatory      3396..3405
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3402..3404,3405..3407,3408..4121)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3447..3468)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3708..3727)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4055..4076
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5506..6094
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5559..5579)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5977..5988)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6169..6402
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6521..6539)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6607..6711
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6712..6780,6781..7572)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6930..6949)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7743..8331
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8232..8251
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8485..8502
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
       61 ttttttggag gcctaggctt ttgcaaaaag ctccccgtgg cacgacaggt ttcccgactg
      121 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      181 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      241 tcacacagga aacagctatg acatgattac gaatttcaca aataaagcat ttttttcact
      301 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcaactg
      361 gataactcaa gctaaccaaa atcatcccaa acttcccacc ccatacccta ttaccactgc
      421 caattacctg tggtttcatt tactctaaac ctgtgattcc tctgaattat tttcatttta
      481 aagaaattgt atttgttaaa tatgtactac aaacttagta gttggaaggg ctaattcact
      541 cccaaagaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg
      601 attagcagaa ctacacacca gggccagggg tcagatatcc actgaccttt ggatggtgct
      661 acaagctagt accagttgag ccagataagg tagaagaggc caataaagga gagaacacca
      721 gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtgttagagt
      781 ggaggtttga cagccgccta gcatttcatc acgtggcccg agagctgcat ccggagtact
      841 tcaagaactg ctgatatcga gcttgctaca agggactttc cgctggggac tttccaggga
      901 ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg
      961 ctttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     1021 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     1081 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     1141 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
     1201 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
     1261 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
     1321 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
     1381 cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac
     1441 gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac
     1501 agctacaacc atcccttcag acaggatcag aagaacttag atcattatat aatacagtag
     1561 caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca
     1621 agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc ggccgctgat
     1681 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     1741 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     1801 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     1861 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     1921 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     1981 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     2041 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     2101 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     2161 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     2221 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     2281 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     2341 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     2401 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     2461 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
     2521 attagtgaac ggatctcgac ggtatcgccg aattcacaaa tggcagtatt catccacaat
     2581 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     2641 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
     2701 gtttattaca gggacagcag agatccagtt tggactagtg gcctccgcgc cgggttttgg
     2761 cgcctcccgc gggcgccccc ctcctcacgg cgagcgctgc cacgtcagac gaagggcgca
     2821 gcgagcgtcc tgatccttcc gcccggacgc tcaggacagc ggcccgctgc tcataagact
     2881 cggccttaga accccagtat cagcagaagg acattttagg acgggacttg ggtgactcta
     2941 gggcactggt tttctttcca gagagcggaa caggcgagga aaagtagtcc cttctcggcg
     3001 attctgcgga gggatctccg tggggcggtg aacgccgatg attatataag gacgcgccgg
     3061 gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg tttgtggatc
     3121 gctgtgatcg tcacttggcg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     3181 atccgctagc gctaccggac tcagatctcg agctcgccac catgattcac accaacctga
     3241 agaaaaagtt cagctgctgc gtcctggtct ttcttctgtt tgcagtcatc tgtgtgtgga
     3301 aggaaaagaa gaaagggagt tactatgatt cctttaaatt gcaaaccaag gaattcctgc
     3361 agtcgacggt accgcgggcc cgggatccac cggtcgccac catggtgagc aagggcgagg
     3421 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3481 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3541 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3601 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3661 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3721 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3781 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3841 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3901 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     3961 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca
     4021 agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     4081 ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc gactctagat
     4141 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     4201 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     4261 ttataatggt aacctacaaa cgggtggagg aacaccccac cctccagaca cattacacag
     4321 aaatccaatc aaacagaagc accatcaggg cttctgctac caaatttatc tcaaaaaact
     4381 acaacaagga atcaccatca gggattccct gtgcaatata cgtcaaacga gggccacgac
     4441 gggaggacga tcacgcctcc cgaatatcgg catgtctggc tttcgaattc agtgcgtgga
     4501 gcatcagccc acgcagccaa tcagagtcga atacaagtcg actttcgcga agagcatcag
     4561 ccttcgcgcc attcttacac aaaccacact ctcccctaca ggaacagcat cagcgttcct
     4621 gcccagtacc caactcaaga aaatttatgt ccccatgcag catcagcgca tgggccccaa
     4681 gaatacatcc ccaacaaaat cacatccgag caccaacagg gctcggagtg ttgtttcttg
     4741 tccaactgga caaaccctcc atggaccatc aggccatgga ctctcaccaa caagacaaaa
     4801 actactcttc tcgaagcagc atcagcgctt cgaaacactc gagcatacat tgtgcctatt
     4861 tcttgggtgg acgatcacgc cacccatgct ctcacgaatt tcaaaacacg gacaaggacg
     4921 agcaccacca gggctcgtcg ttccacgtcc aatacgatta cttacctttc gggatcacga
     4981 tcacggatcc cgcagctaca tcacttccac tcaggacatt caagcatgca cgatcacggc
     5041 atgctccaca agtctcaacc acagaaacta ccaaatgggt tcagcaccag cgaacccact
     5101 cctacctcaa acctcttccc acaaaactgg caagcaggat caccgcttgc ccattccaac
     5161 ataccaaatc aaaaacaatt actggtacag catcagcgta ccagcccaca tctctcacta
     5221 ctatcaaaaa ccaaaccgtt cagcaacagc gaacggtaca cacggaaaaa tcaactggtt
     5281 tacaaatacg aaagacgatc acgctttcgt ccagcgcaaa ctattacgaa aaacatccga
     5341 cgggaagagc aacagccttc ccgcggcgga aaacctcaca aaaacacgac aaacggatgc
     5401 acgaacacgg catccgccga caacccacaa acttacaacc aggcaaacgg tgcaggatca
     5461 ccgcaccgta catcaaacac ctcagatctc atatcgatag atcctaatca acctctggat
     5521 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     5581 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     5641 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     5701 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     5761 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     5821 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     5881 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     5941 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     6001 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     6061 acgagtcgga tctccctttg ggccgcctcc ccgcctgaga tcctttaaga ccaatgactt
     6121 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     6181 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     6241 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     6301 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     6361 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     6421 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggcccgggt
     6481 taattaagga aagggctaga tcattcttga agacgaaagg gcctcgtgat acgcctattt
     6541 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     6601 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     6661 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     6721 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     6781 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     6841 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     6901 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     6961 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     7021 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     7081 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     7141 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     7201 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     7261 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     7321 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     7381 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     7441 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     7501 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     7561 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     7621 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     7681 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7741 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7801 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7861 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     7921 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7981 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     8041 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     8101 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     8161 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     8221 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     8281 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     8341 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     8401 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     8461 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     8521 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagcaa gctcatggct
     8581 gactaatttt tttt
//
