LOCUS       sequence-418716-        5641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses NanoLuc mRNA with the TH in the 3'-UTR.
            Also expresses a hygromycin resistance marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5641)
  AUTHORS   Hatfield BM, Weidmann CA, McCutchin CA, Irving PS, Nelson IM, Felder
            S, Weeks KM
  TITLE     Functional Proximity across an mRNA.
  JOURNAL   Biochemistry. 2025 Sep 16;64(18):3854-3865. doi: 
            10.1021/acs.biochem.5c00340. Epub 2025 Aug 12.
  PUBMED    40792674
REFERENCE   2  (bases 1 to 5641)
  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"; doi: 
            "10.1021/acs.biochem.5c00340"; journalName: "Biochemistry"; date: 
            "2025-09-16- 16"; volume: "64"; issue: "18"; pages: "3854-3865"
COMMENT     Sequence Label: pNL3.2.CMV.3UTR-TH.NLuc-PEST.Hygro
FEATURES             Location/Qualifiers
     source          1..5641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        151..530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        531..734
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     685..705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     731..755
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      853..862
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             859..1371
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     misc_RNA        1539..1557
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     polyA_signal    1596..1717
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1607..1626)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1661..1680
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        1911..2268
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1911..2102
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1934..1954)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2119..2254
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2181..2200
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2299..3336
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             3315..3323
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    3360..3408
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3564..3581)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3735..4323)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3815..3834)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4523..5314,5315..5383))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     polyA_signal    5488..5536
                     /note="synthetic polyadenylation signal"
     misc_feature    5550..5641
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5590..5609
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggcctaactg gcctcaatat tggccattag ccatattatt cattggttat atagcataaa
       61 tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg tacatttata
      121 ttggctcatg tccaatatga ccgccatgtt ggcattgatt attgactagt tattaatagt
      181 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      241 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      301 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      361 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta
      421 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
      481 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      541 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      601 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      661 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      721 atataagcag agctcgttta gtgaaccgtc agatcactag aagctttatt gcggtagttt
      781 atcacagtta aattgctaac gcagtcagtg ggcctcggcg gccaagcttg gcaatccggt
      841 actgttggta aagccaccat ggtcttcaca ctcgaagatt tcgttgggga ctggcgacag
      901 acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag tttgtttcag
      961 aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga aaatgggctg
     1021 aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca aatgggccag
     1081 atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa ggtgatcctg
     1141 cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta tttcggacgg
     1201 ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg
     1261 aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct gctgttccga
     1321 gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc gaattctcac
     1381 ggctttccgc ctgaggttga agagcaagcc gccgatacat tgcctatgtc ctgcgcacaa
     1441 gaaagcggta tggaccggca cccagccgct tgtgcttcag ctcgcatcaa cgtctaaggc
     1501 cgcgactcta gagtcgttct aggcgatcgc tcgagaaaac atgaggatca cccatgtctg
     1561 caggtcgact ctagaaacgc ttcgagcaga catgataaga tacattgatg agtttggaca
     1621 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     1681 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     1741 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     1801 atgtggtaaa atcgataagg atccgtttgc gtattgggcg ctcttccgct gatctgcgca
     1861 gcaccatggc ctgaaataac ctctgaaaga ggaacttggt tagctacctt ctgaggcgga
     1921 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     1981 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2041 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2101 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2161 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     2221 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcgattct
     2281 tctgacacta gcgccaccat gaagaagccc gaactcaccg ctaccagcgt tgaaaaattt
     2341 ctcatcgaga agttcgacag tgtgagcgac ctgatgcagt tgtcggaggg cgaagagagc
     2401 cgagccttca gcttcgatgt cggcggacgc ggctatgtac tgcgggtgaa tagctgcgct
     2461 gatggcttct acaaagaccg ctacgtgtac cgccacttcg ccagcgctgc actacccatc
     2521 cccgaagtgt tggacatcgg cgagttcagc gagagcctga catactgcat cagtagacgc
     2581 gcccaaggcg ttactctcca agacctcccc gaaacagagc tgcctgctgt gttacagcct
     2641 gtcgccgaag ctatggatgc tattgccgcc gccgacctca gtcaaaccag cggcttcggc
     2701 ccattcgggc cccaaggcat cggccagtac acaacctggc gggatttcat ttgcgccatt
     2761 gctgatcccc atgtctacca ctggcagacc gtgatggacg acaccgtgtc cgccagcgta
     2821 gctcaagccc tggacgaact gatgctgtgg gccgaagact gtcccgaggt gcgccacctc
     2881 gtccatgccg acttcggcag caacaacgtc ctgaccgaca acggccgcat caccgccgta
     2941 atcgactggt ccgaagctat gttcggggac agtcagtacg aggtggccaa catcttcttc
     3001 tggcggccct ggctggcttg catggagcag cagactcgct acttcgagcg ccggcatccc
     3061 gagctggccg gcagccctcg tctgcgagcc tacatgctgc gcatcggcct ggatcagctc
     3121 taccagagcc tcgtggacgg caacttcgac gatgctgcct gggctcaagg ccgctgcgat
     3181 gccatcgtcc gcagcggggc cggcaccgtc ggtcgcacac aaatcgctcg ccggagcgca
     3241 gccgtatgga ccgacggctg cgtcgaggtg ctggccgaca gcggcaaccg ccggcccagt
     3301 acacgaccgc gcgctaagga ggtaggtcga gtttaaactc tagaaccggt catggccgca
     3361 ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgtt cgaactagat
     3421 gctgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     3481 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     3541 tgccggcagc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3601 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3661 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3721 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3781 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3841 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3901 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3961 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4021 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4081 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4141 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4201 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4261 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4321 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4381 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4441 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagcggccgc
     4501 aaatgctaaa ccactgcagt ggttaccagt gcttgatcag tgaggcaccg atctcagcga
     4561 tctgcctatt tcgttcgtcc atagtggcct gactccccgt cgtgtagatc actacgattc
     4621 gtgagggctt accatcaggc cccagcgcag caatgatgcc gcgagagccg cgttcaccgg
     4681 cccccgattt gtcagcaatg aaccagccag cagggagggc cgagcgaaga agtggtcctg
     4741 ctactttgtc cgcctccatc cagtctatga gctgctgtcg tgatgctaga gtaagaagtt
     4801 cgccagtgag tagtttccga agagttgtgg ccattgctac tggcatcgtg gtatcacgct
     4861 cgtcgttcgg tatggcttcg ttcaactctg gttcccagcg gtcaagccgg gtcacatgat
     4921 cacccatatt atgaagaaat gcagtcagct ccttagggcc tccgatcgtt gtcagaagta
     4981 agttggccgc ggtgttgtcg ctcatggtaa tggcagcact acacaattct cttaccgtca
     5041 tgccatccgt aagatgcttt tccgtgaccg gcgagtactc aaccaagtcg ttttgtgagt
     5101 agtgtatacg gcgaccaagc tgctcttgcc cggcgtctat acgggacaac accgcgccac
     5161 atagcagtac tttgaaagtg ctcatcatcg ggaatcgttc ttcggggcgg aaagactcaa
     5221 ggatcttgcc gctattgaga tccagttcga tatagcccac tcttgcaccc agttgatctt
     5281 cagcatcttt tactttcacc agcgtttcgg ggtgtgcaaa aacaggcaag caaaatgccg
     5341 caaagaaggg aatgagtgcg acacgaaaat gttggatgct catactcgtc ctttttcaat
     5401 attattgaag catttatcag ggttactagt acgtctctca aggataagta agtaatatta
     5461 aggtacggga ggtattggac aggccgcaat aaaatatctt tattttcatt acatctgtgt
     5521 gttggttttt tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa
     5581 caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc
     5641 t
//
