LOCUS       sequence-81488-e        4477 bp    DNA     circular SYN 14-APR-2025
DEFINITION  serves as PCR-template to generate HR donors for C-terminal GFP-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4477)
  AUTHORS   Bottcher R, Hollmann M, Merk K, Nitschko V, Obermaier C, 
            Philippou-Massier J, Wieland I, Gaul U, Forstemann K
  TITLE     Efficient chromosomal gene modification with CRISPR/cas9 and 
            PCR-based homologous recombination donors in cultured Drosophila 
            cells.
  JOURNAL   Nucleic Acids Res. 2014 Apr 19.
  PUBMED    24748663
REFERENCE   2  (bases 1 to 4477)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2014 Apr 19."
COMMENT     Sequence Label: pMH3
FEATURES             Location/Qualifiers
     source          1..4477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     305..324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        305..323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(358..360,361..363,364..1077)
                     /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(358..360,361..363,364..1074)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(403..424)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(664..683)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1011..1032
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1084..1117)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             1394..1792
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(1841..1874)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        complement(join(2309..2315,2316..2333,2334..2339))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2354..2375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2495..2512)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2666..3254)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2746..2765)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3425..4216,4217..4285))
                     /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     4048..4067
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4286..4390)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4458..4476
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc
       61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc
      121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact
      181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt
      241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc
      301 ggcgtaatac gactcactat agggagagcg gccgccagat cttccggatg gctcgagatg
      361 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      421 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      481 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      541 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      601 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      661 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      721 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      781 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
      841 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
      901 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      961 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1021 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaact
     1081 agtgaagttc ctatactttc tagagaatag gaacttcaaa taaacatatg ctgttggaat
     1141 atactattca acctacaaaa ataacgttaa acaacactac tttatatttg atatgaatgg
     1201 ccacaccttt tatgccataa aacatattgt aagagaatac cactcttttt attccttctt
     1261 tccttcttgt acgttttttg ctgtgagtag gtcgtggtgc tggtgttgca gttgaaataa
     1321 cttaaaatat aaatcataaa actcaaacat aaacttgact atttatttat ttattaagaa
     1381 aggaggtacc ggtatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
     1441 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
     1501 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
     1561 cgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
     1621 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
     1681 tcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     1741 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct agaaattatt
     1801 aaaaataaaa ctttaaaaat aaatttgttt ctaacatatg gaagttccta tactttctag
     1861 agaataggaa cttcatcttt ctagaagatc tcctacaata ttctcagctg ccatggaaaa
     1921 tcgatgttct tcttttattc tctcaagatt ttcaggctgt atattaaaac ttatattaag
     1981 aactatgcta accacctcat caggaaccgt tgtaggtggc gtgggttttc ttggcaatcg
     2041 actctcatga aaactacgag ctaaatattc aatatgttcc tcttgaccaa ctttattctg
     2101 catttttttt gaacgaggtt tagagcaagc ttcaggaaac tgagacagga attttattaa
     2161 aaatttaaat tttgaagaaa gttcagggtt aatagcatcc attttttgct ttgcaagttc
     2221 ctcagcattc ttaacaaaag acgtctcttt tgacatgttt aaagtttaaa cctcctgtgt
     2281 gaaattatta tccgctcata attccacaca ttatacgagc cggaagcata aagtgtaaag
     2341 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgccaattg
     2401 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2461 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2521 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2581 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2641 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2701 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2761 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2821 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2881 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2941 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3001 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3061 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     3121 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3181 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3241 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3301 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3361 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3421 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3481 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3541 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3601 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3661 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3721 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3781 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3841 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3901 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3961 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4021 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4081 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4141 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4201 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4261 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4321 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4381 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     4441 tgacattaac ctataaaaat aggcgtatca cgaggcc
//
