LOCUS       sequence-81489-e        3817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  serves as PCR-template to generate HR donors for C-terminal 
            2xFLAG-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3817)
  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 3817)
  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: "Nucleic 
            Acids Res. 2014 Apr 19."
COMMENT     Sequence Label: pMH4
FEATURES             Location/Qualifiers
     source          1..3817
                     /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             358..381
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             391..414
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(424..457)
                     /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             734..1132
                     /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(1181..1214)
                     /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(1649..1655,1656..1673,1674..1679))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    1694..1715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1835..1852)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2006..2594)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2086..2105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2765..3556,3557..3625))
                     /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     3388..3407
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3626..3730)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3798..3816
                     /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 gctcgaggat
      361 tataaagatg atgatgataa atccggagcc gattataaag atgatgatga taaatgaact
      421 agtgaagttc ctatactttc tagagaatag gaacttcaaa taaacatatg ctgttggaat
      481 atactattca acctacaaaa ataacgttaa acaacactac tttatatttg atatgaatgg
      541 ccacaccttt tatgccataa aacatattgt aagagaatac cactcttttt attccttctt
      601 tccttcttgt acgttttttg ctgtgagtag gtcgtggtgc tggtgttgca gttgaaataa
      661 cttaaaatat aaatcataaa actcaaacat aaacttgact atttatttat ttattaagaa
      721 aggaggtacc ggtatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
      781 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
      841 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
      901 cgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
      961 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
     1021 tcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     1081 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct agaaattatt
     1141 aaaaataaaa ctttaaaaat aaatttgttt ctaacatatg gaagttccta tactttctag
     1201 agaataggaa cttcatcttt ctagaagatc tcctacaata ttctcagctg ccatggaaaa
     1261 tcgatgttct tcttttattc tctcaagatt ttcaggctgt atattaaaac ttatattaag
     1321 aactatgcta accacctcat caggaaccgt tgtaggtggc gtgggttttc ttggcaatcg
     1381 actctcatga aaactacgag ctaaatattc aatatgttcc tcttgaccaa ctttattctg
     1441 catttttttt gaacgaggtt tagagcaagc ttcaggaaac tgagacagga attttattaa
     1501 aaatttaaat tttgaagaaa gttcagggtt aatagcatcc attttttgct ttgcaagttc
     1561 ctcagcattc ttaacaaaag acgtctcttt tgacatgttt aaagtttaaa cctcctgtgt
     1621 gaaattatta tccgctcata attccacaca ttatacgagc cggaagcata aagtgtaaag
     1681 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgccaattg
     1741 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1801 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1861 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1921 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1981 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2041 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2101 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2161 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2221 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2281 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2341 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2401 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     2461 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2521 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2581 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2641 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2701 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2761 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2821 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2881 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     2941 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3001 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3061 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3121 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3181 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3241 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3301 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3361 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3421 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3481 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3541 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3601 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3661 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3721 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     3781 tgacattaac ctataaaaat aggcgtatca cgaggcc
//
