LOCUS       sequence-418742-        5629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses NanoLuc mRNA with no TH as a control. 
            Also expresses a hygromycin resistance marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5629)
  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 5629)
  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.NLuc-PEST.Hygro
FEATURES             Location/Qualifiers
     source          1..5629
                     /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"
     polyA_signal    1584..1705
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1595..1614)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1649..1668
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        1899..2256
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1899..2090
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1922..1942)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2107..2242
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2169..2188
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2287..3324
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             3303..3311
                     /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    3348..3396
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3552..3569)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3723..4311)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3803..3822)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4511..5302,5303..5371))
                     /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    5476..5524
                     /note="synthetic polyadenylation signal"
     misc_feature    5538..5629
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5578..5597
                     /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 gagtcgctcg agggctggta ccgttgctag cacgagcggc cgcttaccgg
     1561 ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1621 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1681 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     1741 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     1801 cgataaggat ccgtttgcgt attgggcgct cttccgctga tctgcgcagc accatggcct
     1861 gaaataacct ctgaaagagg aacttggtta gctaccttct gaggcggaaa gaaccagctg
     1921 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     1981 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2041 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     2101 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2161 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     2221 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcgattcttc tgacactagc
     2281 gccaccatga agaagcccga actcaccgct accagcgttg aaaaatttct catcgagaag
     2341 ttcgacagtg tgagcgacct gatgcagttg tcggagggcg aagagagccg agccttcagc
     2401 ttcgatgtcg gcggacgcgg ctatgtactg cgggtgaata gctgcgctga tggcttctac
     2461 aaagaccgct acgtgtaccg ccacttcgcc agcgctgcac tacccatccc cgaagtgttg
     2521 gacatcggcg agttcagcga gagcctgaca tactgcatca gtagacgcgc ccaaggcgtt
     2581 actctccaag acctccccga aacagagctg cctgctgtgt tacagcctgt cgccgaagct
     2641 atggatgcta ttgccgccgc cgacctcagt caaaccagcg gcttcggccc attcgggccc
     2701 caaggcatcg gccagtacac aacctggcgg gatttcattt gcgccattgc tgatccccat
     2761 gtctaccact ggcagaccgt gatggacgac accgtgtccg ccagcgtagc tcaagccctg
     2821 gacgaactga tgctgtgggc cgaagactgt cccgaggtgc gccacctcgt ccatgccgac
     2881 ttcggcagca acaacgtcct gaccgacaac ggccgcatca ccgccgtaat cgactggtcc
     2941 gaagctatgt tcggggacag tcagtacgag gtggccaaca tcttcttctg gcggccctgg
     3001 ctggcttgca tggagcagca gactcgctac ttcgagcgcc ggcatcccga gctggccggc
     3061 agccctcgtc tgcgagccta catgctgcgc atcggcctgg atcagctcta ccagagcctc
     3121 gtggacggca acttcgacga tgctgcctgg gctcaaggcc gctgcgatgc catcgtccgc
     3181 agcggggccg gcaccgtcgg tcgcacacaa atcgctcgcc ggagcgcagc cgtatggacc
     3241 gacggctgcg tcgaggtgct ggccgacagc ggcaaccgcc ggcccagtac acgaccgcgc
     3301 gctaaggagg taggtcgagt ttaaactcta gaaccggtca tggccgcaat aaaatatctt
     3361 tattttcatt acatctgtgt gttggttttt tgtgtgttcg aactagatgc tgtcgaccga
     3421 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     3481 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     3541 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3601 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3661 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3721 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3781 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3841 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3901 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3961 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4021 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4081 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4141 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4201 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4261 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4321 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4381 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4441 aaatcaatct aaagtatata tgagtaaact tggtctgaca gcggccgcaa atgctaaacc
     4501 actgcagtgg ttaccagtgc ttgatcagtg aggcaccgat ctcagcgatc tgcctatttc
     4561 gttcgtccat agtggcctga ctccccgtcg tgtagatcac tacgattcgt gagggcttac
     4621 catcaggccc cagcgcagca atgatgccgc gagagccgcg ttcaccggcc cccgatttgt
     4681 cagcaatgaa ccagccagca gggagggccg agcgaagaag tggtcctgct actttgtccg
     4741 cctccatcca gtctatgagc tgctgtcgtg atgctagagt aagaagttcg ccagtgagta
     4801 gtttccgaag agttgtggcc attgctactg gcatcgtggt atcacgctcg tcgttcggta
     4861 tggcttcgtt caactctggt tcccagcggt caagccgggt cacatgatca cccatattat
     4921 gaagaaatgc agtcagctcc ttagggcctc cgatcgttgt cagaagtaag ttggccgcgg
     4981 tgttgtcgct catggtaatg gcagcactac acaattctct taccgtcatg ccatccgtaa
     5041 gatgcttttc cgtgaccggc gagtactcaa ccaagtcgtt ttgtgagtag tgtatacggc
     5101 gaccaagctg ctcttgcccg gcgtctatac gggacaacac cgcgccacat agcagtactt
     5161 tgaaagtgct catcatcggg aatcgttctt cggggcggaa agactcaagg atcttgccgc
     5221 tattgagatc cagttcgata tagcccactc ttgcacccag ttgatcttca gcatctttta
     5281 ctttcaccag cgtttcgggg tgtgcaaaaa caggcaagca aaatgccgca aagaagggaa
     5341 tgagtgcgac acgaaaatgt tggatgctca tactcgtcct ttttcaatat tattgaagca
     5401 tttatcaggg ttactagtac gtctctcaag gataagtaag taatattaag gtacgggagg
     5461 tattggacag gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg
     5521 tgtgaatcga tagtactaac atacgctctc catcaaaaca aaacgaaaca aaacaaacta
     5581 gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa catttctct
//
