LOCUS       sequence-477827-        4420 bp    DNA     circular SYN 07-JUL-2025
DEFINITION  Template for in vitro transcription of secreted nanoluciferase, with
            interleukin 6 signal peptide, 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 4420)
  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 4420)
  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: pIL6-Nluc-hbb
FEATURES             Location/Qualifiers
     source          1..4420
                     /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             425..937
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     polyA_signal    1137..1258
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1148..1167)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1202..1221
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1580..1600
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     complement(1752..1769)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1923..2511)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2003..2022)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2682..3473,3474..3542))
                     /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     3305..3324
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3543..3647)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3674..4129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3761..3780)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3971..3992
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4260..4308
                     /note="synthetic polyadenylation signal"
     misc_feature    4322..4413
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4362..4381
                     /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 gactccttct ccacaagcgc
      361 cttcggtcca gttgccttct ccctgggcct gctcctggtg ttgcctgctg ccttccctgc
      421 cccagtcttc acactcgaag atttcgttgg ggactggcga cagacagccg gctacaacct
      481 ggaccaagtc cttgaacagg gaggtgtgtc cagtttgttt cagaatctcg gggtgtccgt
      541 aactccgatc caaaggattg tcctgagcgg tgaaaatggg ctgaagatcg acatccatgt
      601 catcatcccg tatgaaggtc tgagcggcga ccaaatgggc cagatcgaaa aaatttttaa
      661 ggtggtgtac cctgtggatg atcatcactt taaggtgatc ctgcactatg gcacactggt
      721 aatcgacggg gttacgccga acatgatcga ctatttcgga cggccgtatg aaggcatcgc
      781 cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg tggaacggca acaaaattat
      841 cgacgagcgc ctgatcaacc ccgacggctc cctgctgttc cgagtaacca tcaacggagt
      901 gaccggctgg cggctgtgcg aacgcattct ggcgtaagct cgctttcttg ctgtccaatt
      961 tctattaaag gttcctttgt tccctaagtc caactactaa actgggggat attatgaagg
     1021 gccttgagca tctggattct gcctaataaa aaacatttat tttcattgca agaattcccg
     1081 ggcccgcgga tccatgagct agagtcgggg cggccggccg cttcgagcag acatgataag
     1141 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     1201 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     1261 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     1321 aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatctgaacg atggagcgga
     1381 gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg
     1441 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     1501 tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     1561 gagcctgggg actttccaca ccctaactga cacacattcc acagcggatc cgtcgaccga
     1621 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     1681 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     1741 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1801 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1861 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1921 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1981 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2041 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2101 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2161 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2221 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2281 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2341 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2401 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2461 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2521 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2581 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2641 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2701 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2761 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2821 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2881 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2941 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3001 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3061 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3121 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3181 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3241 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3301 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3361 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3421 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3481 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3541 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3601 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3661 aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3721 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3781 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3841 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3901 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3961 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4021 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4081 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttg ccattcgcca
     4141 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     4201 cccaagctac catgataagt aagtaatatt aaggtacggg aggtacttgg agcggccgca
     4261 ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac
     4321 taacatacgc tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc
     4381 ccagtgcaag tgcaggtgcc agaacatttc tctatcgata
//
