LOCUS       sequence-398431-        7185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector with double fluorescence system for targeted 
            integration using CRISPR/Cas9. Expresses 5´HA--eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7185)
  AUTHORS   Hertel O, Neuss A, Busche T, Brandt D, Kalinowski J, Bahnemann J, 
            Noll T
  TITLE     Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated
            site-specific integration into regions with distinct histone 
            modifications.
  JOURNAL   Front Bioeng Biotechnol. 2022 Oct 13;10:1010719. doi: 
            10.3389/fbioe.2022.1010719. eCollection 2022.
  PUBMED    36312557
REFERENCE   2  (bases 1 to 7185)
  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: "Front 
            Bioeng Biotechnol."; date: "2022-10-13"; volume: "10:1010719. doi"; 
            pages: " 10.3389/fbioe.2022.1010719. eCollection 2022"
COMMENT     Sequence Label: pCMV-eGFP-mPlum-HA
FEATURES             Location/Qualifiers
     source          1..7185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        69..372
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        373..576
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      628..637
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(634..636,637..639,640..1353)
                     /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(634..636,637..639,640..1350)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(679..700)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(940..959)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1287..1308
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1371..1388)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1377..1488
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1606..1626)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1611..1940
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1791..1926
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1853..1872
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        1988..2035
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2054..2653
                     /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(2054..2073)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2550..2570
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2783..2904
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        2984..3287
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3288..3491
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3540..4217
                     /codon_start=1
                     /product="far-red monomeric derivative of DsRed fluorescent
                     protein (Wang et al., 2004)"
                     /label=mPlum
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKEHMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     RLKVTKGGPLPFAWDILSPQIMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGEFIYKVKVRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEMK
                     MRLRLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRH
                     STGA"
     primer_bind     3941..3960
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4135..4158
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4337..4458
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(4512..5303,5304..5372))
                     /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     5135..5154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5373..5464)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5506..5525
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5531..5550
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5636..6224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6125..6144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6378..6395
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6493..6510
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6494..6510
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6966..6982
                     /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)."
     misc_feature    join(7097..7185,1..3)
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     7137..7156
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 tctactagtg aattcggtac cgagctcctc gaggggtcat tagttcatag cccatatatg
       61 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      121 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      181 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      241 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      301 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      361 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      421 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      481 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      541 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
      601 gcttactggc ttatggatcc accggtcgcc accatggtga gcaagggcga ggagctgttc
      661 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      721 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      781 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
      841 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      901 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      961 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1021 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1081 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1141 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1201 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     1261 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1321 atcactctcg gcatggacga gctgtacaag taaagcggcc gcaaaatcag cctcgactgt
     1381 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1441 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcac aacactcaac
     1501 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1561 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     1621 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     1681 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     1741 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     1801 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     1861 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     1921 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     1981 agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     2041 gaggaactaa accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     2101 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     2161 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     2221 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     2281 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     2341 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     2401 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     2461 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     2521 gcccgccttc ctggagacat ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     2581 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     2641 gcccggtgcc tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt
     2701 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     2761 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     2821 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     2881 gtccaaactc atcaatgtat cttatcatgt ctgtatagct cgtagattta attaaacgcg
     2941 tgcgatcgca aggccgtcaa ggccgcatat atatggagtt ccgcgttaca taacttacgg
     3001 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     3061 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     3121 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     3181 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     3241 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     3301 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     3361 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     3421 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     3481 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtgg ctacccacca
     3541 tggtgagcaa gggcgaggag gtcatcaagg agttcatgcg cttcaaggag cacatggagg
     3601 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     3661 gcacccagac cgccaggctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     3721 tcctgtcccc tcagatcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     3781 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     3841 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     3901 acaaggtgaa ggtgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     3961 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     4021 agatgaagat gaggctgagg ctgaaggacg gcggccacta cgacgccgag gtcaagacca
     4081 cctacatggc caagaagccc gtgcagctgc ccggcgccta caagaccgac atcaagctgg
     4141 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc gccgagggcc
     4201 gccactccac cggcgccttc gaagataact gatcataatc agccatacca catttgtaga
     4261 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     4321 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4381 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4441 actcatcaat gtatcttact gggcctcatg ggccgtcgac ctctagctag agcttggcgt
     4501 aatcatggtc attaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4561 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4621 ccatctggcc ccagcgctgc gatgataccg cgagaaccac gctcaccggc tccggattta
     4681 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4741 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4801 agtttgcgca acgttgttgc catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4861 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4921 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4981 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5041 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5101 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5161 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5221 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5281 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5341 ataagggcga cacggaaatg ttgaatactc atattcttcc tttttcaata ttattgaagc
     5401 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5461 caaatagggg tcagtgttac aaccaattaa ccaattctga acattatcgc gagcccattt
     5521 atacctgaat atggctcata acaccccttg ctcatgacca aaatccctta acgtgagtta
     5581 cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5641 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5701 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5761 gagcgcagat accaaatact gttcttctag tgtagccgta gttagcccac cacttcaaga
     5821 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5881 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5941 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6001 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6061 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6121 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6181 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6241 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6301 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6361 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaggcgag agtagggaac
     6421 tgccaggcat caaactaagc agaaggcccc tgacggatgg cctttttgcg tttctacaaa
     6481 ctctttctgt gttgtaaaac gacggccagt cttaagctcg ggccccctgg gcggttctga
     6541 taacgagtaa tcgttaatcc gcaaataacg taaaaacccg cttcggcggg tttttttatg
     6601 gggggagttt agggaaagag catttgtcag aatatttaag ggcgcctgtc actttgcttg
     6661 atatatgaga attatttaac cttataaatg agaaaaaagc aacgcacttt aaataagata
     6721 cgttgctttt tcgattgatg aacacctata attaaactat tcatctatta tttatgattt
     6781 tttgtatata caatatttct agtttgttaa agagaattaa gaaaataaat ctcgaaaata
     6841 ataaagggaa aatcagtttt tgatatcaaa attatacatg tcaacgataa tacaaaatat
     6901 aatacaaact ataagatgtt atcagtattt attatcattt agaataaatt ttgtgtcgcc
     6961 cttaattgtg agcggataac aattacgagc ttcatgcaca gtggcgttga cattgattat
     7021 tgactagcat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     7081 tgcattagtt attaataaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag
     7141 caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttc
//
