LOCUS       sequence-477838-        4336 bp    DNA     circular SYN 07-JUL-2025
DEFINITION  Template for in vitro transcription of nanoluciferase 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 4336)
  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 4336)
  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: pNluc-hbb
FEATURES             Location/Qualifiers
     source          1..4336
                     /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             338..853
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    1053..1174
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1064..1083)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1118..1137
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1496..1516
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     complement(1668..1685)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1839..2427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1919..1938)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2598..3389,3390..3458))
                     /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     3221..3240
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3459..3563)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3590..4045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3677..3696)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3887..3908
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4176..4224
                     /note="synthetic polyadenylation signal"
     misc_feature    4238..4329
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4278..4297
                     /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 gtcttcacac tcgaagattt
      361 cgttggggac tggcgacaga cagccggcta caacctggac caagtccttg aacagggagg
      421 tgtgtccagt ttgtttcaga atctcggggt gtccgtaact ccgatccaaa ggattgtcct
      481 gagcggtgaa aatgggctga agatcgacat ccatgtcatc atcccgtatg aaggtctgag
      541 cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca
      601 tcactttaag gtgatcctgc actatggcac actggtaatc gacggggtta cgccgaacat
      661 gatcgactat ttcggacggc cgtatgaagg catcgccgtg ttcgacggca aaaagatcac
      721 tgtaacaggg accctgtgga acggcaacaa aattatcgac gagcgcctga tcaaccccga
      781 cggctccctg ctgttccgag taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg
      841 cattctggcg taagctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc
      901 taagtccaac tactaaactg ggggatatta tgaagggcct tgagcatctg gattctgcct
      961 aataaaaaac atttattttc attgcaagaa ttcccgggcc cgcggatcca tgagctagag
     1021 tcggggcggc cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     1081 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     1141 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     1201 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     1261 tggtaaaatc gataaggatc tgaacgatgg agcggagaat gggcggaact gggcggagtt
     1321 aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg
     1381 ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa
     1441 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct
     1501 aactgacaca cattccacag cggatccgtc gaccgatgcc cttgagagcc ttcaacccag
     1561 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
     1621 ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac
     1681 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1741 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1801 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1861 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1921 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1981 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2041 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2101 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2161 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2221 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     2281 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2341 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2401 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2461 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2521 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2581 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2641 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2701 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     2761 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2821 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     2881 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     2941 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3001 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3061 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3121 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3181 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3241 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3301 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3361 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3421 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3481 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3541 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt
     3601 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3661 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3721 tttccccgtc aagctctaaa tctggggctc cctttagggt tccgatttag tgctttacgg
     3781 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3841 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3901 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3961 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4021 aacaaaatat taacgcttac aatttgccat tcgccattca ggctgcgcaa ctgttgggaa
     4081 gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg ataagtaagt
     4141 aatattaagg tacgggaggt acttggagcg gccgcaataa aatatcttta ttttcattac
     4201 atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc catcaaaaca
     4261 aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa
     4321 catttctcta tcgata
//
