LOCUS       sequence-136541-        4673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  template for N-terminal eGFP tag, inducible Blasticidin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4673)
  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 4673)
  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: pRB30
FEATURES             Location/Qualifiers
     source          1..4673
                     /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             complement(453..851)
                     /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(1254..1287)
                     /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(1312..1314,1315..1317,1318..2028)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1312..1314,1315..1317,1318..2028)
                     /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(1357..1378)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1618..1637)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1965..1986
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(join(2505..2511,2512..2529,2530..2535))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2550..2571
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2691..2708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2862..3450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2942..2961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3621..4412,4413..4481))
                     /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     4244..4263
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4482..4586)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4654..4672
                     /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 ttagaaacaa
      421 atttattttt aaagttttat ttttaataat ttctagccct cccacacata accagagggc
      481 agcaattcac gaatcccaac tgccgtcggc tgtccatcac tgtccttcac tatggctttg
      541 atcccaggat gcagatcgag aagaacctgt cggcaccgtc cgcaggggct caagatgccc
      601 ctgttctcat ttccgatcgc gacgatacaa gtcaggttgc cagctgccgc agcagcagca
      661 gtgcccagca ccacgagttc tgcgcaaggt cccccagtaa aatgatatac attgacacca
      721 gtgaagatgc ggccgtcgct agagagagct gcgctggcga cgctgtagtc ttcagagatg
      781 gggatgctgt tgattgtagc cgttgctctt tcaatgaggg tggattcttc ttgagacaaa
      841 ggcttggcca tatgcctagg cttgttgatt tgttttttgt ttatttactg cggtaattcc
      901 tagtcttaga agcttattga acttgttgat gccttctggt tgggaaatcc tcctctttat
      961 acccgatcca gtccaatcga aactgtgggc cgtggtgccc gataagataa gaaagccgtg
     1021 tgcaagtcga tcggcgtgcg caaaagcata atgctttttc tgcccggcga tgataagcga
     1081 tttgcacacg ccctgataac aggaattgat ttttttggtg cgcaaagcag tgaaagctct
     1141 ataagaatgc tggaccgcca gtcgacaatc atcaaacagt gctagctaac ggctaagtga
     1201 atcaagttta ttttaatata atcaacaaca aaatatttta acacaaacct agggaagttc
     1261 ctatactttc tagagaatag gaacttctaa tccaaaatgg gatccaccgg tatggtgagc
     1321 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1381 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1441 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1501 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1561 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1621 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1681 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1741 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1801 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1861 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1921 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1981 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga attcggtacc
     2041 tctccagctg ctccaagcgg cggtactagt atctttctag aagatctcct acaatattct
     2101 cagctgccat ggaaaatcga tgttcttctt ttattctctc aagattttca ggctgtatat
     2161 taaaacttat attaagaact atgctaacca cctcatcagg aaccgttgta ggtggcgtgg
     2221 gttttcttgg caatcgactc tcatgaaaac tacgagctaa atattcaata tgttcctctt
     2281 gaccaacttt attctgcatt ttttttgaac gaggtttaga gcaagcttca ggaaactgag
     2341 acaggaattt tattaaaaat ttaaattttg aagaaagttc agggttaata gcatccattt
     2401 tttgctttgc aagttcctca gcattcttaa caaaagacgt ctcttttgac atgtttaaag
     2461 tttaaacctc ctgtgtgaaa ttattatccg ctcataattc cacacattat acgagccgga
     2521 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2581 cgctcactgc caattgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2641 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2701 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2761 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2821 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2881 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2941 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3001 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3061 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3121 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3181 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3241 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3301 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3361 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3421 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3481 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3541 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3601 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3661 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3721 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3781 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3841 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3901 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3961 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4021 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4081 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4141 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4201 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4261 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4321 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4381 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4441 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4501 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4561 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     4621 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gcc
//
