LOCUS       sequence-136547-        4933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  template for N-terminal eGFP tag, constitutive Blasticidin 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4933)
  AUTHORS   Kunzelmann S, Bottcher R, Schmidts I, Forstemann K
  TITLE     A Comprehensive Toolbox for Genome Editing in Cultured Drosophila 
            melanogaster Cells.
  JOURNAL   G3 (Bethesda). 2016 Jun 1;6(6):1777-85. doi: 10.1534/g3.116.028241.
  PUBMED    27172193
REFERENCE   2  (bases 1 to 4933)
  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: "10.1534/g3.116"; 
            journalName: "G3 (Bethesda)"; date: "2016-06-1- 1"; volume: "6"; 
            issue: "6"; pages: "1777-85"
COMMENT     Sequence Label: pRB40
FEATURES             Location/Qualifiers
     source          1..4933
                     /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"
     protein_bind    complement(372..404)
                     /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             674..1072
                     /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(1520..1553)
                     /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             join(1572..1574,1575..1577,1578..2288)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1572..1574,1575..1577,1578..2288)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1617..1638)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1878..1897)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2225..2246
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(join(2765..2771,2772..2789,2790..2795))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2810..2831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2951..2968)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3122..3710)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3202..3221)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3881..4672,4673..4741))
                     /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     4504..4523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4742..4846)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4914..4932
                     /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 gctcgagttt
      361 ttcagcaaga taagttccta tactttctag agaataggaa cttccatatg ctgttggaat
      421 atactattca acctacaaaa ataacgttaa acaacactac tttatatttg atatgaatgg
      481 ccacaccttt tatgccataa aacatattgt aagagaatac cactcttttt attccttctt
      541 tccttcttgt acgttttttg ctgtgagtag gtcgtggtgc tggtgttgca gttgaaataa
      601 cttaaaatat aaatcataaa actcaaacat aaacttgact atttatttat ttattaagaa
      661 aggaggtacc ggtatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
      721 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
      781 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
      841 cgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
      901 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
      961 tcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     1021 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct agaaattatt
     1081 aaaaataaaa ctttaaaaat aaatttgttt ctaacatatg cctaggcttg ttgatttgtt
     1141 ttttgtttat ttactgcggt aattcctagt cttagaagct tattgaactt gttgatgcct
     1201 tctggttggg aaatcctcct ctttataccc gatccagtcc aatcgaaact gtgggccgtg
     1261 gtgcccgata agataagaaa gccgtgtgca agtcgatcgg cgtgcgcaaa agcataatgc
     1321 tttttctgcc cggcgatgat aagcgatttg cacacgccct gataacagga attgattttt
     1381 ttggtgcgca aagcagtgaa agctctataa gaatgctgga ccgccagtcg acaatcatca
     1441 aacagtgcta gctaacggct aagtgaatca agtttatttt aatataatca acaacaaaat
     1501 attttaacac aaacctaggg aagttcctat actttctaga gaataggaac ttctaatcca
     1561 aaatgggatc catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1621 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1681 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1741 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1801 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1861 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1921 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1981 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2041 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2101 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2161 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2221 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2281 tgtacaagga attcggtacc tctccagctg ctccaagcgg cggtactagt atctttctag
     2341 aagatctcct acaatattct cagctgccat ggaaaatcga tgttcttctt ttattctctc
     2401 aagattttca ggctgtatat taaaacttat attaagaact atgctaacca cctcatcagg
     2461 aaccgttgta ggtggcgtgg gttttcttgg caatcgactc tcatgaaaac tacgagctaa
     2521 atattcaata tgttcctctt gaccaacttt attctgcatt ttttttgaac gaggtttaga
     2581 gcaagcttca ggaaactgag acaggaattt tattaaaaat ttaaattttg aagaaagttc
     2641 agggttaata gcatccattt tttgctttgc aagttcctca gcattcttaa caaaagacgt
     2701 ctcttttgac atgtttaaag tttaaacctc ctgtgtgaaa ttattatccg ctcataattc
     2761 cacacattat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2821 aactcacatt aattgcgttg cgctcactgc caattgcttt ccagtcggga aacctgtcgt
     2881 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2941 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3001 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3061 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3121 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3181 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3241 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3301 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3361 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3421 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3481 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3541 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     3601 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3661 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3721 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3781 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3841 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3901 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3961 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4021 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4081 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4141 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4201 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4261 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4321 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4381 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4441 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4501 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4561 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4621 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4681 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4741 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4801 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4861 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4921 gtatcacgag gcc
//
