LOCUS       sequence-477837-        4405 bp    DNA     circular SYN 07-JUL-2025
DEFINITION  Template for in vitro transcription of secreted nanoluciferase, with
            signal peptide from PMEL, flanked by the human haemoglobin beta 5' 
            and 3' UTRs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4405)
  AUTHORS   Smart AD, Hughes ME, Ruiz Velasco AD, Hori N, Stolnik S, Jopling CL
  TITLE     A proximity labelling-based approach to directly detect mRNA 
            delivery to specific subcellular locations
  JOURNAL   Molecular Therapy Nucleic Acids, 102602. doi: 
            10.1016/j.omtn.2025.102602
REFERENCE   2  (bases 1 to 4405)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Molecular 
            Therapy Nucleic Acids, 102602. doi: 10.1016/j.omtn.2025.102602"
COMMENT     Sequence Label: pPMEL-Nluc-hbb
FEATURES             Location/Qualifiers
     source          1..4405
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        48..244
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      95..230
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     268..287
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        268..286
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             410..922
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     polyA_signal    1122..1243
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1133..1152)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1187..1206
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1565..1585
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     complement(1737..1754)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1908..2496)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1988..2007)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2667..3458,3459..3527))
                     /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     3290..3309
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3528..3632)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3659..4114
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3746..3765)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3956..3977
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4245..4293
                     /note="synthetic polyadenylation signal"
     misc_feature    4307..4398
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4347..4366
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgcgatctgc atctcaatta
       61 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
      121 cgcccattct ccgccccatc gctgactaat tttttttatt tatgcagagg ccgaggccgc
      181 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
      241 caaaaagctt acttggtcag cggccgctaa tacgactcac tatagggaca tttgcttctg
      301 acacaactgt gttcactagc aacctcaaac agacaccatg gatctggtgc taaaaagatg
      361 ccttcttcat ttggctgtga taggtgcttt gctggctgtg ggggctacag tcttcacact
      421 cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc aagtccttga
      481 acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc cgatccaaag
      541 gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca tcccgtatga
      601 aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt
      661 ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg acggggttac
      721 gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa
      781 aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg agcgcctgat
      841 caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg gctggcggct
      901 gtgcgaacgc attctggcgt aagctcgctt tcttgctgtc caatttctat taaaggttcc
      961 tttgttccct aagtccaact actaaactgg gggatattat gaagggcctt gagcatctgg
     1021 attctgccta ataaaaaaca tttattttca ttgcaagaat tcccgggccc gcggatccat
     1081 gagctagagt cggggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt
     1141 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     1201 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     1261 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     1321 ctacaaatgt ggtaaaatcg ataaggatct gaacgatgga gcggagaatg ggcggaactg
     1381 ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg
     1441 agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt
     1501 gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt
     1561 ccacacccta actgacacac attccacagc ggatccgtcg accgatgccc ttgagagcct
     1621 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     1681 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg
     1741 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1801 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1861 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     1921 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1981 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2041 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2101 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2161 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2221 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2281 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2341 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2401 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2461 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2521 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2581 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2641 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2701 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2761 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2821 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2881 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2941 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3001 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3061 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3121 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3181 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3241 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3301 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3361 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3421 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3481 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3541 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3601 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3661 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3721 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3781 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3841 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3901 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3961 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4021 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4081 gcgaatttta acaaaatatt aacgcttaca atttgccatt cgccattcag gctgcgcaac
     4141 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagcccaa gctaccatga
     4201 taagtaagta atattaaggt acgggaggta cttggagcgg ccgcaataaa atatctttat
     4261 tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc
     4321 atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag
     4381 gtgccagaac atttctctat cgata
//
