LOCUS       sequence-345032-        7885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nLuc with 5' leader of CoV2 in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7885)
  AUTHORS   Mendez AS, Ly M, Gonzalez-Sanchez AM, Hartenian E, Ingolia NT, Cate 
            JH, Glaunsinger BA
  TITLE     The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in 
            suppression of cellular gene expression and preservation of viral 
            gene expression.
  JOURNAL   Cell Rep. 2021 Oct 19;37(3):109841. doi: 
            10.1016/j.celrep.2021.109841. Epub 2021 Sep 30.
  PUBMED    34624207
REFERENCE   2  (bases 1 to 7885)
  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"; journalName: "Cell Rep.";
            date: "2021-10-19"; volume: "37(3):109841. doi"; pages: " 
            10.1016/j.celrep.2021.109841. Epub 2021 Sep 30"
COMMENT     Sequence Label: pJP-CoV2leader-nLuc-TSS
FEATURES             Location/Qualifiers
     source          1..7885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    95..116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(131..136,137..154,155..161)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    169..185
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     174..196
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     193..209
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     193..209
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     228..248
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        230..248
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        276..502
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             503..683
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    730..855
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1348..1581
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1766..1810
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     primer_bind     2108..2128
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        2417..2620
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2571..2591
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2697..3212
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     misc_feature    3254..3371
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3420..3930
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3849..3867
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3952..4551
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3952..3971)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4448..4468
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4679..4912
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4984..5105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5021..5040)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5075..5094
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5145..5280
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5300..5319)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5301..5319)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5329..5346)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5329..5345)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5338..5360)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5487..5942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5574..5593)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5784..5805
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5968..6072
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6073..6141,6142..6933)
                     /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     complement(6291..6310)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7104..7692
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7593..7612
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7846..7863
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
       61 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      121 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      181 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      241 actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta
      301 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg
      361 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac
      421 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa
      481 gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct
      541 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
      601 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
      661 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa
      721 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag
      781 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat
      841 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg
      901 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag
      961 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
     1021 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
     1081 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct
     1141 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct
     1201 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     1261 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     1321 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     1381 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     1441 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     1501 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     1561 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     1621 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     1681 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     1741 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     1801 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     1861 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     1921 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     1981 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat
     2041 tcgattagtg aacggatctc gacggtatcg atcacgagac tagcctcgag cggccgcccc
     2101 cttcaccgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     2161 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     2221 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     2281 ctatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     2341 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     2401 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     2461 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     2521 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     2581 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgaccttc
     2641 ccaggtaaca aaccaaccaa ctttcgatct cttgtagatc tgttctctaa acgaacatgg
     2701 tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc
     2761 aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc
     2821 cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca
     2881 tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg
     2941 tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg
     3001 acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt
     3061 tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg
     3121 agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg
     3181 gctggcggct gtgcgaacgc attctggcgt aggaattctc gacctcgaga caaatggcag
     3241 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     3301 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     3361 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg
     3421 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct
     3481 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc
     3541 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct
     3601 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg
     3661 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg
     3721 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc
     3781 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg
     3841 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc
     3901 gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag
     3961 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     4021 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     4081 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     4141 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     4201 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     4261 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     4321 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     4381 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     4441 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     4501 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc
     4561 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga
     4621 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     4681 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     4741 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     4801 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     4861 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt
     4921 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     4981 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5041 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5101 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     5161 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     5221 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     5281 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     5341 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     5401 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     5461 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     5521 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     5581 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5641 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5701 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5761 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     5821 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     5881 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     5941 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     6001 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     6061 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     6121 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6181 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6241 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     6301 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6361 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6421 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6481 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6541 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6601 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6661 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6721 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6781 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6841 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6901 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6961 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     7021 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     7081 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     7141 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     7201 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     7261 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     7321 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     7381 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7441 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7501 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7561 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7621 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7681 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7741 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     7801 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     7861 gaggaagcgg aagagcgccc aatac
//
