LOCUS       sequence-336026-        6034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA donor plasmid for site-specific integration of 
            IRES-Puro-pEF1a-EGFP cassette at the ACTB locus using 
            Cas9-transposase fusion proteins..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6034)
  AUTHORS   Ma S, Wang X, Hu Y, Lv J, Liu C, Liao K, Guo X, Wang D, Lin Y, Rong 
            Z
  TITLE     Enhancing site-specific DNA integration by a Cas9 nuclease fused 
            with a DNA donor-binding domain.
  JOURNAL   Nucleic Acids Res. 2020 Oct 9;48(18):10590-10601. doi: 
            10.1093/nar/gkaa779.
  PUBMED    32986839
REFERENCE   2  (bases 1 to 6034)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2020-10-9- 9"; volume: "48"; issue: 
            "18"; pages: "10590-10601"
COMMENT     Sequence Label: pBlue-ACTB-IR-IRES-Puro-pEF1-alpha-EGFP-IR-ACTB
FEATURES             Location/Qualifiers
     source          1..6034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..212
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      219..228
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(225..227,228..230,231..944)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(225..227,228..230,231..941)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(270..291)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(531..550)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     878..899
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    951..1032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(988..1007)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     polyA_signal    1039..1263
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1252..1269
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1301..1900)
                     /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(1384..1404)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1881..1900
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     misc_feature    join(1901..1909,1910..1912,1913..2487)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2074..2093)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     2303..2320
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(2844..2863)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2845..2863)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2873..2890)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2873..2889)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2882..2904)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3031..3486
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3118..3137)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3328..3349
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3512..3616
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3617..3685,3686..4477)
                     /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(3835..3854)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4648..5236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5137..5156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5390..5407
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5524..5545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5560..5565,5566..5583,5584..5590)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5598..5614
                     /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     5603..5625
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5622..5638
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5622..5638
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5657..5677
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5659..5677
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat
       61 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      121 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      181 tttttcgcaa cgggtttgcc gccagaacac agctcgaggc caccatggtg agcaagggcg
      241 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
      301 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
      361 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
      421 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
      481 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
      541 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
      601 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
      661 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
      721 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
      781 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
      841 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
      901 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaagatatc aacttgttta
      961 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     1021 ttttttcact gcctcgaccc atagagccca ccgcatcccc agcatgcctg ctattgtctt
     1081 cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac
     1141 tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt
     1201 ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca
     1261 cagtcgaggc tgatcagcgg gtttaaacgg gccctctaga tcaggcaccg ggcttgcggg
     1321 tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga
     1381 gccgctcgta gaaggggagg ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg
     1441 cgcgctcggc cgcctccact ccggggagca cgacggcgct gcccagaccc ttgccctggt
     1501 ggtcgggcga gacgccgacg gtggccagga accacgcggg ctccttgggc cggtgcggcg
     1561 ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga accgctcaac tcggccatgc
     1621 gcgggccgat ctcggcgaac accgcccccg cttcgacgct ctccggcgtg gtccagaccg
     1681 ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga
     1741 ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg gtccggatcg acggtgtggc
     1801 gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg tacggccctg gggacgtcgt
     1861 cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat ggttgtggcc atattatcat
     1921 cgtgtttttc aaaggaaaac cacgtccccg tggttcgggg ggcctagacg tttttttaac
     1981 ctcgactaaa cacatgtaaa gcatgtgcac cgaggcccca gatcagatcc catacaatgg
     2041 ggtaccttct gggcatcctt cagccccttg ttgaatacgc ttgaggagag ccatttgact
     2101 ctttccacaa ctatccaact cacaacgtgg cactggggtt gtgccgcctt tgcaggtgta
     2161 tcttatacac gtggcttttg gccgcagagg cacctgtcgc caggtggggg gttccgctgc
     2221 ctgcaaaggg tcgctacaga cgttgtttgt cttcaagaag cttccagagg aactgcttcc
     2281 ttcacgacat tcaacagacc ttgcattcct ttggcgagag gggaaagacc cctaggaatg
     2341 ctcgtcaaga agacagggcc aggtttccgg gccctcacat tgccaaaaga cggcaatatg
     2401 gtggaaaata acatatagac aaacgcacac cggccttatt ccaagcggct tcggccagta
     2461 acgttagggg ggggggaggg agaggggcgg aattaagctt ctaaagccat gacatcattt
     2521 tctggaattt tccaagctgt ttaaaggcac agtcaactta gtgtatgtaa acttctgacc
     2581 cactggaatt gtgatacagt gaattataag tgaaataatc tgtctgtaaa caattgttgg
     2641 aaaaatgact tgtgtcatgc acaaagtaga tgtcctaact gacttgccaa aactattgtt
     2701 tgttaacaag aaatttgtgg agtagttgaa aaacgagttt taatgactcc aacttaagtg
     2761 tatgtaaact tccgacttca actgtaaccg gtccaaatat gagatgcgtt gttacctcga
     2821 gggggggccc ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     2881 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2941 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     3001 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     3061 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3121 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3181 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3241 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3301 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3361 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     3421 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     3481 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3541 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3601 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3661 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3721 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3781 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3841 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3901 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3961 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4021 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4081 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4141 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4201 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4261 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4321 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4381 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4441 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4501 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4561 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4621 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4681 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4741 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4801 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4861 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4921 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4981 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5041 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5101 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5161 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5221 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5281 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5341 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5401 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     5461 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     5521 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     5581 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     5641 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctccaccg
     5701 cggtggccca aatatgagat gcgttgttac gctagctaca gttgaagtcg gaagtttaca
     5761 tacacttaag ttggagtcat taaaactcgt ttttcaacta ctccacaaat ttcttgttaa
     5821 caaacaatag ttttggcaag tcagttagga catctacttt gtgcatgaca caagtcattt
     5881 ttccaacaat tgtttacaga cagattattt cacttataat tcactgtatc acaattccag
     5941 tgggtcagaa gtttacatac actaagttga ctgtgccttt aaacagcttg gaaaattcca
     6001 gaaaatgatg tcatggcttt agaagcttac tagt
//
