LOCUS       sequence-418738-        5690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses NanoLuc mRNA with the TH in the 5'-UTR.
            Also expresses a hygromycin resistance marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5690)
  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 5690)
  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.5UTR-TH.NLuc-PEST.Hygro
FEATURES             Location/Qualifiers
     source          1..5690
                     /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"
     misc_RNA        811..829
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     regulatory      914..923
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             920..1432
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    1645..1766
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1656..1675)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1710..1729
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        1960..2317
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1960..2151
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1983..2003)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2168..2303
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2230..2249
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2348..3385
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             3364..3372
                     /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    3409..3457
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3613..3630)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3784..4372)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3864..3883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4572..5363,5364..5432))
                     /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    5537..5585
                     /note="synthetic polyadenylation signal"
     misc_feature    5599..5690
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5639..5658
                     /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 atcacagttt ctaggcgatc gctcgagaaa acatgaggat cacccatgtc tgcaggtcga
      841 ctctagaaat aaattgctaa cgcagtcagt gggcctcggc ggccaagctt ggcaatccgg
      901 tactgttggt aaagccacca tggtcttcac actcgaagat ttcgttgggg actggcgaca
      961 gacagccggc tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgtttca
     1021 gaatctcggg gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg aaaatgggct
     1081 gaagatcgac atccatgtca tcatcccgta tgaaggtctg agcggcgacc aaatgggcca
     1141 gatcgaaaaa atttttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct
     1201 gcactatggc acactggtaa tcgacggggt tacgccgaac atgatcgact atttcggacg
     1261 gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctgtg
     1321 gaacggcaac aaaattatcg acgagcgcct gatcaacccc gacggctccc tgctgttccg
     1381 agtaaccatc aacggagtga ccggctggcg gctgtgcgaa cgcattctgg cgaattctca
     1441 cggctttccg cctgaggttg aagagcaagc cgccgataca ttgcctatgt cctgcgcaca
     1501 agaaagcggt atggaccggc acccagccgc ttgtgcttca gctcgcatca acgtctaagg
     1561 ccgcgactct agagtcgctc gagggctggt accgttgcta gcacgagcgg ccgcttaccg
     1621 gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta
     1681 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     1741 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     1801 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
     1861 tcgataagga tccgtttgcg tattgggcgc tcttccgctg atctgcgcag caccatggcc
     1921 tgaaataacc tctgaaagag gaacttggtt agctaccttc tgaggcggaa agaaccagct
     1981 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2041 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2101 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     2161 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     2221 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     2281 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcgattctt ctgacactag
     2341 cgccaccatg aagaagcccg aactcaccgc taccagcgtt gaaaaatttc tcatcgagaa
     2401 gttcgacagt gtgagcgacc tgatgcagtt gtcggagggc gaagagagcc gagccttcag
     2461 cttcgatgtc ggcggacgcg gctatgtact gcgggtgaat agctgcgctg atggcttcta
     2521 caaagaccgc tacgtgtacc gccacttcgc cagcgctgca ctacccatcc ccgaagtgtt
     2581 ggacatcggc gagttcagcg agagcctgac atactgcatc agtagacgcg cccaaggcgt
     2641 tactctccaa gacctccccg aaacagagct gcctgctgtg ttacagcctg tcgccgaagc
     2701 tatggatgct attgccgccg ccgacctcag tcaaaccagc ggcttcggcc cattcgggcc
     2761 ccaaggcatc ggccagtaca caacctggcg ggatttcatt tgcgccattg ctgatcccca
     2821 tgtctaccac tggcagaccg tgatggacga caccgtgtcc gccagcgtag ctcaagccct
     2881 ggacgaactg atgctgtggg ccgaagactg tcccgaggtg cgccacctcg tccatgccga
     2941 cttcggcagc aacaacgtcc tgaccgacaa cggccgcatc accgccgtaa tcgactggtc
     3001 cgaagctatg ttcggggaca gtcagtacga ggtggccaac atcttcttct ggcggccctg
     3061 gctggcttgc atggagcagc agactcgcta cttcgagcgc cggcatcccg agctggccgg
     3121 cagccctcgt ctgcgagcct acatgctgcg catcggcctg gatcagctct accagagcct
     3181 cgtggacggc aacttcgacg atgctgcctg ggctcaaggc cgctgcgatg ccatcgtccg
     3241 cagcggggcc ggcaccgtcg gtcgcacaca aatcgctcgc cggagcgcag ccgtatggac
     3301 cgacggctgc gtcgaggtgc tggccgacag cggcaaccgc cggcccagta cacgaccgcg
     3361 cgctaaggag gtaggtcgag tttaaactct agaaccggtc atggccgcaa taaaatatct
     3421 ttattttcat tacatctgtg tgttggtttt ttgtgtgttc gaactagatg ctgtcgaccg
     3481 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     3541 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     3601 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3661 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3721 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3781 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3841 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3901 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3961 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4021 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4081 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4141 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4201 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4261 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4321 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4381 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4441 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4501 taaatcaatc taaagtatat atgagtaaac ttggtctgac agcggccgca aatgctaaac
     4561 cactgcagtg gttaccagtg cttgatcagt gaggcaccga tctcagcgat ctgcctattt
     4621 cgttcgtcca tagtggcctg actccccgtc gtgtagatca ctacgattcg tgagggctta
     4681 ccatcaggcc ccagcgcagc aatgatgccg cgagagccgc gttcaccggc ccccgatttg
     4741 tcagcaatga accagccagc agggagggcc gagcgaagaa gtggtcctgc tactttgtcc
     4801 gcctccatcc agtctatgag ctgctgtcgt gatgctagag taagaagttc gccagtgagt
     4861 agtttccgaa gagttgtggc cattgctact ggcatcgtgg tatcacgctc gtcgttcggt
     4921 atggcttcgt tcaactctgg ttcccagcgg tcaagccggg tcacatgatc acccatatta
     4981 tgaagaaatg cagtcagctc cttagggcct ccgatcgttg tcagaagtaa gttggccgcg
     5041 gtgttgtcgc tcatggtaat ggcagcacta cacaattctc ttaccgtcat gccatccgta
     5101 agatgctttt ccgtgaccgg cgagtactca accaagtcgt tttgtgagta gtgtatacgg
     5161 cgaccaagct gctcttgccc ggcgtctata cgggacaaca ccgcgccaca tagcagtact
     5221 ttgaaagtgc tcatcatcgg gaatcgttct tcggggcgga aagactcaag gatcttgccg
     5281 ctattgagat ccagttcgat atagcccact cttgcaccca gttgatcttc agcatctttt
     5341 actttcacca gcgtttcggg gtgtgcaaaa acaggcaagc aaaatgccgc aaagaaggga
     5401 atgagtgcga cacgaaaatg ttggatgctc atactcgtcc tttttcaata ttattgaagc
     5461 atttatcagg gttactagta cgtctctcaa ggataagtaa gtaatattaa ggtacgggag
     5521 gtattggaca ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt
     5581 gtgtgaatcg atagtactaa catacgctct ccatcaaaac aaaacgaaac aaaacaaact
     5641 agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga acatttctct
//
