LOCUS       sequence-418744-        5650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses NanoLuc mRNA with the TH in the CDS2 
            position. Also expresses a hygromycin resistance marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5650)
  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 5650)
  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.CDS2-TH.NLuc-PEST.Hygro
FEATURES             Location/Qualifiers
     source          1..5650
                     /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"
     misc_RNA        1263..1281
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     polyA_signal    1605..1726
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1616..1635)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1670..1689
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        1920..2277
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1920..2111
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1943..1963)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2128..2263
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2190..2209
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2308..3345
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             3324..3332
                     /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    3369..3417
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3573..3590)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3744..4332)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3824..3843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4532..5323,5324..5392))
                     /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    5497..5545
                     /note="synthetic polyadenylation signal"
     misc_feature    5559..5650
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5599..5618
                     /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 gtacatgagg atcacccatg taacggcaac aaaattatcg acgagcgcct gatcaacccc
     1321 gacggctccc tgctgttccg agtaaccatc aacggagtga ccggctggcg gctgtgcgaa
     1381 cgcattctgg cgaattctca cggctttccg cctgaggttg aagagcaagc cgccgataca
     1441 ttgcctatgt cctgcgcaca agaaagcggt atggaccggc acccagccgc ttgtgcttca
     1501 gctcgcatca acgtctaagg ccgcgactct agagtcgctc gagggctggt accgttgcta
     1561 gcacgagcgg ccgcttaccg gccggccgct tcgagcagac atgataagat acattgatga
     1621 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1681 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1741 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     1801 cctctacaaa tgtggtaaaa tcgataagga tccgtttgcg tattgggcgc tcttccgctg
     1861 atctgcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt agctaccttc
     1921 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     1981 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2041 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2101 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2161 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     2221 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2281 ctcgattctt ctgacactag cgccaccatg aagaagcccg aactcaccgc taccagcgtt
     2341 gaaaaatttc tcatcgagaa gttcgacagt gtgagcgacc tgatgcagtt gtcggagggc
     2401 gaagagagcc gagccttcag cttcgatgtc ggcggacgcg gctatgtact gcgggtgaat
     2461 agctgcgctg atggcttcta caaagaccgc tacgtgtacc gccacttcgc cagcgctgca
     2521 ctacccatcc ccgaagtgtt ggacatcggc gagttcagcg agagcctgac atactgcatc
     2581 agtagacgcg cccaaggcgt tactctccaa gacctccccg aaacagagct gcctgctgtg
     2641 ttacagcctg tcgccgaagc tatggatgct attgccgccg ccgacctcag tcaaaccagc
     2701 ggcttcggcc cattcgggcc ccaaggcatc ggccagtaca caacctggcg ggatttcatt
     2761 tgcgccattg ctgatcccca tgtctaccac tggcagaccg tgatggacga caccgtgtcc
     2821 gccagcgtag ctcaagccct ggacgaactg atgctgtggg ccgaagactg tcccgaggtg
     2881 cgccacctcg tccatgccga cttcggcagc aacaacgtcc tgaccgacaa cggccgcatc
     2941 accgccgtaa tcgactggtc cgaagctatg ttcggggaca gtcagtacga ggtggccaac
     3001 atcttcttct ggcggccctg gctggcttgc atggagcagc agactcgcta cttcgagcgc
     3061 cggcatcccg agctggccgg cagccctcgt ctgcgagcct acatgctgcg catcggcctg
     3121 gatcagctct accagagcct cgtggacggc aacttcgacg atgctgcctg ggctcaaggc
     3181 cgctgcgatg ccatcgtccg cagcggggcc ggcaccgtcg gtcgcacaca aatcgctcgc
     3241 cggagcgcag ccgtatggac cgacggctgc gtcgaggtgc tggccgacag cggcaaccgc
     3301 cggcccagta cacgaccgcg cgctaaggag gtaggtcgag tttaaactct agaaccggtc
     3361 atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgttc
     3421 gaactagatg ctgtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     3481 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     3541 taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3601 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3661 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3721 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3781 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3841 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3901 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3961 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4021 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4081 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4141 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4201 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4261 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4321 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4381 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4441 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4501 agcggccgca aatgctaaac cactgcagtg gttaccagtg cttgatcagt gaggcaccga
     4561 tctcagcgat ctgcctattt cgttcgtcca tagtggcctg actccccgtc gtgtagatca
     4621 ctacgattcg tgagggctta ccatcaggcc ccagcgcagc aatgatgccg cgagagccgc
     4681 gttcaccggc ccccgatttg tcagcaatga accagccagc agggagggcc gagcgaagaa
     4741 gtggtcctgc tactttgtcc gcctccatcc agtctatgag ctgctgtcgt gatgctagag
     4801 taagaagttc gccagtgagt agtttccgaa gagttgtggc cattgctact ggcatcgtgg
     4861 tatcacgctc gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg tcaagccggg
     4921 tcacatgatc acccatatta tgaagaaatg cagtcagctc cttagggcct ccgatcgttg
     4981 tcagaagtaa gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta cacaattctc
     5041 ttaccgtcat gccatccgta agatgctttt ccgtgaccgg cgagtactca accaagtcgt
     5101 tttgtgagta gtgtatacgg cgaccaagct gctcttgccc ggcgtctata cgggacaaca
     5161 ccgcgccaca tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct tcggggcgga
     5221 aagactcaag gatcttgccg ctattgagat ccagttcgat atagcccact cttgcaccca
     5281 gttgatcttc agcatctttt actttcacca gcgtttcggg gtgtgcaaaa acaggcaagc
     5341 aaaatgccgc aaagaaggga atgagtgcga cacgaaaatg ttggatgctc atactcgtcc
     5401 tttttcaata ttattgaagc atttatcagg gttactagta cgtctctcaa ggataagtaa
     5461 gtaatattaa ggtacgggag gtattggaca ggccgcaata aaatatcttt attttcatta
     5521 catctgtgtg ttggtttttt gtgtgaatcg atagtactaa catacgctct ccatcaaaac
     5581 aaaacgaaac aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga
     5641 acatttctct
//
