LOCUS       sequence-237585-        7771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses puro-2A-BFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7771)
  AUTHORS   Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y, 
            Nakano K, Saitoh N, Liu Z, Yamamoto T, Okamura T, Ohkawa Y, Kimura 
            H, Nikaido I
  TITLE     Genome-wide kinetic properties of transcriptional bursting in mouse 
            embryonic stem cells.
  JOURNAL   Sci Adv. 2020 Jun 17;6(25):eaaz6699. doi: 10.1126/sciadv.aaz6699. 
            eCollection 2020 Jun.
  PUBMED    32596448
REFERENCE   2  (bases 1 to 7771)
  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: "Sci Adv."; 
            date: "2020-06-17"; volume: "6(25):eaaz6699. doi"; pages: " 
            10.1126/sciadv.aaz6699. eCollection 2020 Jun"
COMMENT     Sequence Label: pKLV-PGKpuro2ABFP
FEATURES             Location/Qualifiers
     source          1..7771
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        715..1018
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1020..1218
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1173..1193
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1236..1416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1463..1588
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2085..2318
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2503..2547
                     /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    2845..2962
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3063..3562
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3098..3121)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3495..3514
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3602..4198
                     /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(3602..3621)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4098..4118
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4217..4270
                     /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             4271..4972
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(5327..5344)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5333..5557
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5583..5603)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5583..5601)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5622..5638)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5622..5638)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5635..5657)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5646..5662
                     /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(5670..5676,5677..5694,5695..5700))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5715..5736
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5853..5870)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6024..6612)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6104..6123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6783..7574,7575..7643))
                     /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     7406..7425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7644..7748)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgactagtta
      661 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      721 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      781 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      841 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      901 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      961 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1021 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     1081 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     1141 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     1201 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct
     1261 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     1321 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     1381 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     1441 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     1501 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     1561 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     1621 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     1681 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     1741 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1801 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1861 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1921 gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     1981 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     2041 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     2101 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     2161 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     2221 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     2281 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     2341 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     2401 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     2461 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     2521 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     2581 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     2641 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     2701 ctcccaaccc cgaggggacc caacaggccc gaaggaatag aagaagaagg tggagagaga
     2761 gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag
     2821 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     2881 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     2941 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt
     3001 agtaccgggc ccgagggcct agaattcaga gctagaaata gcaagttgga tccaattcta
     3061 ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct
     3121 gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag
     3181 gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt
     3241 caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc
     3301 acgtctcact agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct
     3361 ttggggcagc ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag
     3421 gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc
     3481 ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc
     3541 ttcctcatct ccgggccttt cgacctgcat ccatctagat ctcgagcagc tgaagcttac
     3601 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     3661 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     3721 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     3781 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     3841 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     3901 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     3961 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     4021 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     4081 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     4141 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg
     4201 cggcgggtcc ggaggagagg gcagaggaag tctcctaaca tgcggtgacg tggaggagaa
     4261 tcctggccca atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     4321 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     4381 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     4441 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
     4501 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     4561 ggacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     4621 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     4681 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     4741 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     4801 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     4861 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     4921 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt gagcggccgc
     4981 taggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     5041 gaaaaggggg gactggaagg gctaattcac tcccaaagaa gtcaagatct gctttttgcc
     5101 tgtactgggt ctctctggtt agaccagagt ctctctggtt agaccagatc tgagcctggg
     5161 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     5221 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     5281 tttagtcagt gtggaaaatc tctagcagtt taaacccgct gatcagcctc gactgtgcct
     5341 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     5401 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     5461 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagtc
     5521 aatagcaggc atgctgggga tgcggtgggc tctatgggcg gccgttaatt aagcttttgt
     5581 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     5641 tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat aaagtgtaaa
     5701 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5761 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5821 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5881 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5941 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6001 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6061 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6121 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6181 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6241 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6301 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6361 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6421 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6481 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6541 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6601 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6661 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6721 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6781 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6841 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6901 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6961 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7021 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7081 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7141 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7201 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7261 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7321 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7381 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7441 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7501 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7561 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7621 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7681 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7741 gttccgcgca catttccccg aaaagtgcca c
//
