LOCUS       sequence-345040-        7908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nLuc with 5' leader of human beta globin (HBB) in 
            mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7908)
  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 7908)
  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-HBB-nLuc
FEATURES             Location/Qualifiers
     source          1..7908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     97..116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     350..367
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    484..505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(520..525,526..543,544..550)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    558..574
                     /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     563..585
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     582..598
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     582..598
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     617..637
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        619..637
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        665..891
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             892..1072
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1119..1244
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1737..1970
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2155..2199
                     /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     2497..2517
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        2806..3009
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2960..2980
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3091..3100
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3097..3612
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     misc_feature    3666..3783
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3832..4342
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4261..4279
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4364..4963
                     /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(4364..4383)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4860..4880
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5091..5324
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5396..5517
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5433..5452)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5487..5506
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5557..5692
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5712..5731)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5713..5731)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5741..5758)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5741..5757)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5750..5772)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5899..6354
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5986..6005)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6196..6217
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6380..6484
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6485..6553,6554..7345)
                     /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(6703..6722)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7516..7908,1..196)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
       61 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
      121 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      181 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
      241 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
      301 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
      361 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
      421 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      481 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      541 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      601 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
      661 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
      721 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      781 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      841 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      901 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      961 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     1021 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
     1081 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
     1141 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
     1201 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
     1261 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
     1321 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
     1381 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
     1441 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
     1501 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
     1561 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
     1621 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1681 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1741 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1801 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1861 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1921 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1981 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     2041 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     2101 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     2161 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     2221 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     2281 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     2341 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa
     2401 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtatcga
     2461 tcacgagact agcctcgagc ggccgccccc ttcaccgagg gcctatttcc catgattcct
     2521 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     2581 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     2641 gcagttttaa aattatgttt taaaatggac tatcatatgc caagtacgcc ccctattgac
     2701 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     2761 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     2821 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     2881 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     2941 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     3001 agcagagctg gtttagtgaa ccgtcagatc cgctagcgct acatttgctt ctgacacaac
     3061 tgtgttcact agcaacctca aacagacacc gccaccatgg tcttcacact cgaagatttc
     3121 gttggggact ggcgacagac agccggctac aacctggacc aagtccttga acagggaggt
     3181 gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc cgatccaaag gattgtcctg
     3241 agcggtgaaa atgggctgaa gatcgacatc catgtcatca tcccgtatga aggtctgagc
     3301 ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat
     3361 cactttaagg tgatcctgca ctatggcaca ctggtaatcg acggggttac gccgaacatg
     3421 atcgactatt tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact
     3481 gtaacaggga ccctgtggaa cggcaacaaa attatcgacg agcgcctgat caaccccgac
     3541 ggctccctgc tgttccgagt aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc
     3601 attctggcgt aagatatctc tagagaattc tcgacctcga gacaaatggc agtattcatc
     3661 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     3721 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     3781 tttcgggttt attacaggga cagcagagat ccactttggc cgcggctcga gggggttggg
     3841 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg
     3901 ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag
     3961 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc
     4021 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc
     4081 acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc
     4141 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga
     4201 aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg
     4261 gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg
     4321 aatcaccgac ctctctcccc agggggatcc accggagctt accatgaccg agtacaagcc
     4381 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     4441 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     4501 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     4561 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     4621 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     4681 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     4741 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     4801 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     4861 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     4921 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc
     4981 gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg gtacctttaa gaccaatgac
     5041 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     5101 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
     5161 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     5221 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     5281 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca
     5341 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     5401 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5461 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5521 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     5581 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     5641 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     5701 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     5761 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     5821 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     5881 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5941 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     6001 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     6061 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     6121 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     6181 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     6241 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     6301 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     6361 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6421 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     6481 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6541 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6601 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6661 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6721 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6781 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6841 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6901 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6961 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     7021 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     7081 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     7141 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     7201 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     7261 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     7321 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     7381 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     7441 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     7501 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     7561 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     7621 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     7681 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     7741 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     7801 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     7861 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagct
//
