LOCUS       sequence-488053-        7855 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Express Tev-3xFlag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7855)
  AUTHORS   Zhu X, Cruz VE, Zhang H, Erzberger JP, Mendell JT
  TITLE     Specific tRNAs promote mRNA decay by recruiting the CCR4-NOT complex
            to translating ribosomes.
  JOURNAL   Science. 2024 Nov 22;386(6724):eadq8587. doi: 
            10.1126/science.adq8587. Epub 2024 Nov 22.
  PUBMED    39571015
REFERENCE   2  (bases 1 to 7855)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2024-11-22"; volume: "386(6724):eadq8587. doi"; pages: " 
            10.1126/science.adq8587. Epub 2024 Nov 22"
COMMENT     Sequence Label: lenti-CMV-TEV-3XFlag
FEATURES             Location/Qualifiers
     source          1..7855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    139..160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(175..180,181..198,199..205)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    213..229
                     /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     218..240
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     237..253
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     237..253
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     272..292
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        274..292
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        320..546
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             547..727
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    774..899
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1392..1625
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1810..1854
                     /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"
     misc_feature    2121..2237
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2260..2563
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2564..2767
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2718..2738
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2764..2788
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2800..2809
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2809..2829
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2830..2895
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(2948..2972)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3003..3591
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3056..3076)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3474..3485)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3642..3662)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3647..3976
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3827..3962
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4004..4603
                     /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(4004..4023)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4500..4520
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4693..4926
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4998..5119
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5035..5054)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5089..5108
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5159..5294
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5314..5333)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5315..5333)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5343..5360)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5343..5359)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5352..5374)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5501..5956
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5588..5607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5798..5819
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5982..6086
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6087..6155,6156..6947)
                     /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(6305..6324)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7118..7706
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7607..7626
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
       61 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
      121 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
      181 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
      241 aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa
      301 gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg caacatggta
      361 acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg ccgattggtg
      421 gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct gacatggatt
      481 ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct agctcgatac
      541 ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
      601 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
      661 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
      721 tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc
      781 gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga
      841 gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca
      901 gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa
      961 agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag
     1021 ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac
     1081 catcccttca gacaggatca gaagaactta gatcattata taatacagta gcaaccctct
     1141 attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg
     1201 aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga
     1261 ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt
     1321 gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga
     1381 gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc
     1441 gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag
     1501 cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg
     1561 ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag
     1621 ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat
     1681 gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg
     1741 gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac
     1801 cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat
     1861 tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc
     1921 ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga
     1981 tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa
     2041 ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga
     2101 tctcgacggt atcggttaac ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     2161 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     2221 acaaaattca aaattttatc gataagcttg ggagttccgc gttacataac ttacggtaaa
     2281 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     2341 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     2401 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     2461 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2521 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     2581 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     2641 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     2701 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     2761 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgg ccaccatgga gaacctgtac
     2821 ttccagggcg actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag
     2881 gatgacgatg acaagtagaa acactaggat ccggtaccga attcgcggcc gcactcgaga
     2941 tatctagacc cagctttctt gtacaaagtg gttgatatcc agcacagtgg cggccgctcg
     3001 acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3061 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     3121 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     3181 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     3241 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     3301 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3361 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     3421 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     3481 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     3541 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggaattc
     3601 tgcagatatc cggttagtaa tgagtttgga attaattctg tggaatgtgt gtcagttagg
     3661 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3721 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3781 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3841 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3901 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3961 cctaggcttt tgcaaaaagc tcccggatcc accggagctt accatgaccg agtacaagcc
     4021 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     4081 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     4141 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     4201 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     4261 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     4321 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     4381 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     4441 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     4501 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     4561 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgataagcac aattcgagct
     4621 cggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     4681 aaaagggggg actggaaggg ctagctcact cccaacgaag acaagatctg ctttttgctt
     4741 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     4801 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     4861 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     4921 ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     4981 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5041 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5101 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     5161 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     5221 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     5281 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     5341 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     5401 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     5461 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     5521 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     5581 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5641 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5701 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5761 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5821 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     5881 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5941 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     6001 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     6061 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     6121 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6181 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6241 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6301 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6361 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6421 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6481 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6541 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6601 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6661 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6721 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6781 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6841 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6901 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6961 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     7021 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     7081 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     7141 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     7201 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7261 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7321 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7381 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7441 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7501 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7561 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7621 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7681 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7741 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7801 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accga
//
