LOCUS       sequence-81490-e        3850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  serves as PCR-template to generate HR donors for C-terminal 
            Twin-Strep tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3850)
  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 3850)
  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: pIW1
FEATURES             Location/Qualifiers
     source          1..3850
                     /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             364..387
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             424..447
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    complement(457..490)
                     /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             767..1165
                     /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(1214..1247)
                     /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(1682..1688,1689..1706,1707..1712))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    1727..1748
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1868..1885)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2039..2627)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2119..2138)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2798..3589,3590..3658))
                     /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     3421..3440
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3659..3763)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3831..3849
                     /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 gctcgagagc
      361 gcttggagcc acccgcagtt cgagaaaggt ggaggttccg gaggtggatc gggaggtgga
      421 tcgtggagcc acccgcagtt cgaaaaatga actagtgaag ttcctatact ttctagagaa
      481 taggaacttc aaataaacat atgctgttgg aatatactat tcaacctaca aaaataacgt
      541 taaacaacac tactttatat ttgatatgaa tggccacacc ttttatgcca taaaacatat
      601 tgtaagagaa taccactctt tttattcctt ctttccttct tgtacgtttt ttgctgtgag
      661 taggtcgtgg tgctggtgtt gcagttgaaa taacttaaaa tataaatcat aaaactcaaa
      721 cataaacttg actatttatt tatttattaa gaaaggaggt accggtatgg ccaagccttt
      781 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat
      841 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg
      901 tgtcaatgta tatcatttta ctgggggacc ttgcgcagaa ctcgtggtgc tgggcactgc
      961 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg
     1021 catcttgagc ccctgcggac ggtgccgaca ggttcttctc gatctgcatc ctgggatcaa
     1081 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc
     1141 ctctggttat gtgtgggagg gctagaaatt attaaaaata aaactttaaa aataaatttg
     1201 tttctaacat atggaagttc ctatactttc tagagaatag gaacttcatc tttctagaag
     1261 atctcctaca atattctcag ctgccatgga aaatcgatgt tcttctttta ttctctcaag
     1321 attttcaggc tgtatattaa aacttatatt aagaactatg ctaaccacct catcaggaac
     1381 cgttgtaggt ggcgtgggtt ttcttggcaa tcgactctca tgaaaactac gagctaaata
     1441 ttcaatatgt tcctcttgac caactttatt ctgcattttt tttgaacgag gtttagagca
     1501 agcttcagga aactgagaca ggaattttat taaaaattta aattttgaag aaagttcagg
     1561 gttaatagca tccatttttt gctttgcaag ttcctcagca ttcttaacaa aagacgtctc
     1621 ttttgacatg tttaaagttt aaacctcctg tgtgaaatta ttatccgctc ataattccac
     1681 acattatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     1741 tcacattaat tgcgttgcgc tcactgccaa ttgctttcca gtcgggaaac ctgtcgtgcc
     1801 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1861 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1921 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1981 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2041 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2101 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2161 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2221 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2281 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2341 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2401 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2461 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2521 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2581 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2641 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2701 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2761 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2821 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2881 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2941 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3001 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3061 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3121 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3181 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3241 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3301 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3361 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3421 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3481 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3541 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3601 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3661 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3721 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3781 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     3841 tcacgaggcc
//
