LOCUS       sequence-408804-        7830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express puromycin resistance marker and mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7830)
  AUTHORS   Horns F, Martinez JA, Fan C, Haque M, Linton JM, Tobin V, Santat L, 
            Maggiolo AO, Bjorkman PJ, Lois C, Elowitz MB
  TITLE     Engineering RNA export for measurement and manipulation of living 
            cells.
  JOURNAL   Cell. 2023 Jul 10:S0092-8674(23)00689-X. doi: 
            10.1016/j.cell.2023.06.013.
  PUBMED    37437570
REFERENCE   2  (bases 1 to 7830)
  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. 2023 
            Jul 10:S0092-8674(23)00689-X. doi: 10.1016/j.cell.2023.06.013."
COMMENT     Sequence Label: pFH2.155_LV-Puro-T2A-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..7830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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    1807..1923
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1966..2177
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2190..2458
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2514..3110
                     /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(2514..2533)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3010..3030
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3120..3173
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3186..3893
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     misc_feature    3909..4497
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3962..3982)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4380..4391)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4571..4804
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4876..4997
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4913..4932)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4967..4986
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5037..5172
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5205..5221)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5205..5221)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5218..5240)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5229..5245
                     /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)."
     promoter        complement(join(5253..5259,5260..5277,5278..5283))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5298..5319
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5436..5453)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5607..6195)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6366..7157,7158..7226))
                     /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     6989..7008
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7227..7331)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7399..7417
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7455..7477)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7577..7596
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7790..7812
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7804..7821
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7805..7821
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccact gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     1801 gttaactttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     1861 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aattcaaaat
     1921 tttatcgata ctagtggatc tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat
     1981 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt gcctagagaa
     2041 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     2101 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     2161 ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca cgcgcccgcc
     2221 gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg
     2281 gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg agaccgggcc
     2341 tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg ctttgcctga
     2401 ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt tacagatcca
     2461 agctgtgacc ggcgcctacg ctagcgttta aacttaagct tggtaccgcc accatgaccg
     2521 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc
     2581 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgacccg gaccgccaca
     2641 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     2701 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     2761 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     2821 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     2881 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     2941 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3001 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3061 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggatcgggag
     3121 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccaccggtcg
     3181 ccaccgtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag ctgtacatgg
     3241 agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg
     3301 agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
     3361 acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca
     3421 tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
     3481 acgaggacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     3541 tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga
     3601 aaacactcgg ctgggaggcc ttcaccgaaa cgctgtaccc cgctgacggc ggcctggaag
     3661 gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca aacgccaaga
     3721 ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg
     3781 actacagact ggaaagaatc aaggaggcca acaacgaaac ctacgtcgag cagcacgagg
     3841 tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt aattagtcta
     3901 gagggcccaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3961 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     4021 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     4081 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     4141 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     4201 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     4261 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     4321 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     4381 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     4441 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg
     4501 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     4561 aaggggggac tggaagggct aattcactcc caacgaagat aagatctgct ttttgcttgt
     4621 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     4681 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     4741 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     4801 agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     4861 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4921 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4981 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     5041 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5101 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     5161 tttttggagg cctagacttt tgcagagacc aaattcgtaa tcatgtcata gctgtttcct
     5221 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5281 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5341 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5401 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5461 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5521 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5581 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5641 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5701 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5761 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5821 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5881 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5941 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6001 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6061 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6121 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6181 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6241 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6301 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6361 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6421 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6481 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6541 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6601 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6661 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6721 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6781 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6841 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6901 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6961 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7021 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7081 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7141 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7201 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7261 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7321 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     7381 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     7441 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     7501 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     7561 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     7621 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     7681 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     7741 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     7801 cgttgtaaaa cgacggccag tgccaagctg
//
