LOCUS       sequence-233516-        7773 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 7773)
  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 7773)
  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..7773
                     /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    2087..2320
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2505..2549
                     /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    2847..2964
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3065..3564
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3100..3123)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3497..3516
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3604..4200
                     /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(3604..3623)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4100..4120
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4219..4272
                     /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             4273..4974
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(5329..5346)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5335..5559
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5585..5605)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5585..5603)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5624..5640)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5624..5640)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5637..5659)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5648..5664
                     /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(5672..5678,5679..5696,5697..5702))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5717..5738
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5855..5872)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6026..6614)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6106..6125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6785..7576,7577..7645))
                     /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     7408..7427
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7646..7750)
                     /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 ggccgcgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1981 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     2041 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     2101 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     2161 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     2221 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     2281 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     2341 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     2401 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     2461 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     2521 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     2581 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     2641 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     2701 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     2761 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     2821 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     2881 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     2941 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     3001 ttagtaccgg gcccgagggc ctagaattca gagctagaaa tagcaagttg gatccaattc
     3061 taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg
     3121 ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt
     3181 aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta
     3241 gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta
     3301 gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc
     3361 ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga
     3421 aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg
     3481 tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc
     3541 tcttcctcat ctccgggcct ttcgacctgc atccatctag atctcgagca gctgaagctt
     3601 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     3661 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     3721 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     3781 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     3841 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     3901 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     3961 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     4021 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     4081 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     4141 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     4201 ggcggcgggt ccggaggaga gggcagagga agtctcctaa catgcggtga cgtggaggag
     4261 aatcctggcc caatgagcga gctgattaag gagaacatgc acatgaagct gtacatggag
     4321 ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
     4381 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac
     4441 atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc
     4501 cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac
     4561 gaggacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
     4621 tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa
     4681 acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc
     4741 agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc
     4801 acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac
     4861 tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg
     4921 gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa ttgagcggcc
     4981 gctaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     5041 aagaaaaggg gggactggaa gggctaattc actcccaaag aagtcaagat ctgctttttg
     5101 cctgtactgg gtctctctgg ttagaccaga gtctctctgg ttagaccaga tctgagcctg
     5161 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     5221 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     5281 cttttagtca gtgtggaaaa tctctagcag tttaaacccg ctgatcagcc tcgactgtgc
     5341 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     5401 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     5461 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     5521 tcaatagcag gcatgctggg gatgcggtgg gctctatggg cggccgttaa ttaagctttt
     5581 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     5641 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5701 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5761 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5821 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5881 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5941 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     6001 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6061 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6121 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6181 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6241 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6301 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6361 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6421 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     6481 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6541 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6601 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6661 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6721 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6781 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6841 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6901 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6961 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7021 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7081 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7141 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7201 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7261 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7321 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7381 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7441 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7501 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7561 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7621 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7681 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7741 gggttccgcg cacatttccc cgaaaagtgc cac
//
