LOCUS       sequence-373357-        7003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Puromycin-selectable expression of GFP in Drosophila S2 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7003)
  AUTHORS   Kingston ER, Blodgett LW, Bartel DP
  TITLE     Endogenous transcripts direct microRNA degradation in Drosophila, 
            and this targeted degradation is required for proper embryonic 
            development.
  JOURNAL   Mol Cell. 2022 Sep 15. pii: S1097-2765(22)00849-8. doi: 
            10.1016/j.molcel.2022.08.029.
  PUBMED    36150386
REFERENCE   2  (bases 1 to 7003)
  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: "Mol Cell. 
            2022 Sep 15. pii: S1097-2765(22)00849-8. doi: 
            10.1016/j.molcel.2022.08.029."
COMMENT     Sequence Label: HA-GFP_rescue_construct
FEATURES             Location/Qualifiers
     source          1..7003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1640..1666
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1679..1705
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1718..1744
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1808..1810,1811..2521)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1808..1810,1811..2521)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1850..1871)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2111..2130)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2458..2479
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2564..2617
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2642..3241
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2642..2661)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3138..3158
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3249..3383
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3273..3292)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3327..3346
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3984..4572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4064..4083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4743..5534,5535..5603))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5366..5385
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5604..5708)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5776..5794
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_RNA        complement(5946..6021)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ggtctgtggc tgggcgcgac gtcaccgacg ttggagccac tcctttgacc atgtgtgcgt
       61 gtgtgtatta ttcgtgtctc gccactcgcc ggttgttttt ttctttttat gctgcgctct
      121 ctctagcgcc atctcgctta cgcatgctca acgcaccgca tgttgccgtt tccttttatg
      181 cgtcattttg gctcgaaata ggcaattatt taaacaaaga ttagtcaacg aaaacgctaa
      241 aataaataag tctacaatat ggttacttat tgccatgtgt gtgcagccaa cgatagcaac
      301 aaaagcaaca acacaggtgg ctttccctct ttcacttttt gtttgcaagc cgcgtgcgag
      361 caagacggca cgaccggcaa acgcaattac gctgacaaag agcagacgaa gttttggcga
      421 aaaacatcaa ggcgcctgat acgaatgcat ttgcaataac aattgcgata tttaatattg
      481 tttatgaagc tgtttgactt caaaacacac aaaaaaaaaa ataaaacaaa ttatttgaaa
      541 gagaattagg aatcggacgc ttatcgttag ggtaacaaca agaaatgctt actgagtcac
      601 agcctctgga aaactgccgc aagccagaga gagagagaaa aagagggaga gcagcttaga
      661 ccgcatgtgc ttgtgtgtga ggcgtctctc tcttcgtctc tgttgcgcaa acgcatagac
      721 tgcactgaaa aaatcgatta cctatttttt atgaatgaat atttgcacta ttactattca
      781 aaactattaa gatagcaatc acattcaata gccaaatact ataccacctg agcgatgcaa
      841 cgaaatgatc aatttgagca aaaatgctgc atatttagga cggcatcatt atagaaatgc
      901 ttcttgctgt gtacttttct ctcgtctggc agctgtttcg ccgttattgt taaaaccggc
      961 ttaagttagg tgtgttttct acgactagtg aatgccctac tagaagatgt gtgttgcaca
     1021 aaatgtccct ggaataacca atttgaagtg cagatagcag taaacgtaag ctaatatgaa
     1081 tattatttaa ctgtaatgtt ttaatatcgc tggacattac taataaaccc actataaaca
     1141 catgtacata tgtatgtttt ggcatacaat gagtagttgg ggaaaaaatg tgtaaaagca
     1201 ccgtgaccat cacagcataa agataaccag ctgaagtatc gaatatgagt aacccccaaa
     1261 ttgaatcaca tgccgcaact gataggaccc atggaagtac actcttcatg gcgatataca
     1321 agacacacac aagcacgaac acccagttgc ggaggaaatt ctccgtaaat gaaaacccaa
     1381 tcggcgaaca attcataccc atatatggta aaagttttga acgcgacttg agagcggaga
     1441 gcattgcggc tgataaggtt ttagcgctaa gcgggcttta taaaacgggc tgcgggacca
     1501 gttttcatat cactaccgtt tgagttcttg tgctgtgtgg atactcctcc cgacacaaag
     1561 ccgctccatc agccagcagt cgtctaatcc agagaccccg gatcggggta cctactagtc
     1621 cagtgtggtg gaattcatgt atccatatga tgttccagat tatgctggag gttcttcata
     1681 cccctatgat gtgcctgact acgctggcag cgccggatac ccttatgatg tgcctgatta
     1741 tgctggaggg agcggcggaa gtgggagttc aggaggctcc ggatcaagcg gcagtagtgg
     1801 tggaagcgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1861 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1921 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1981 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     2041 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     2101 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     2161 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     2221 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     2281 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     2341 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     2401 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     2461 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     2521 gaaggccggc caggcaaaaa agaaaaaggg atcaggcaag cttgagggca gaggaagtct
     2581 tctaacatgc ggtgacgtgg aggagaatcc cggccctgct agcggtagcg gcagcggtag
     2641 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt
     2701 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga
     2761 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     2821 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     2881 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     2941 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     3001 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     3061 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     3121 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     3181 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     3241 actcgaggga tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     3301 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     3361 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     3421 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg
     3481 attatgatca gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
     3541 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
     3601 ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg
     3661 acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc
     3721 gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg
     3781 gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc
     3841 attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc
     3901 aggcaggtag atgacgacca tcagggacag cttcaaggcc agcaaaaggc caggaaccgt
     3961 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4021 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4081 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4141 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4201 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4261 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4321 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4381 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     4441 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4501 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4561 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4621 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4681 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4741 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4801 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4861 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4921 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4981 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5041 aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5101 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5161 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5221 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5281 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5341 tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg
     5401 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5461 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5521 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5581 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5641 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5701 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     5761 acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attaattcta
     5821 tattctaaaa acacaaatga tacttctaaa aaaaaatcat gaatggcatc aactctgaat
     5881 caaatctttg cagatgcacc tacttctcat ttccactgtc acatcatttt tccagatcta
     5941 aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct tattttaact
     6001 tgctatttct agctctaaaa ctacataacg aataaaaaag gatttaagtg caaatggtaa
     6061 aaaagtgtcg agtttttctt gagttgattg tgctgtaatg agactctgca ttcggcattt
     6121 gactcggctt ttcctactcg tgccgtattt caggctgcaa gtcgaacaga tctcgctgcc
     6181 tgttatgtgg cccacaaacc aagacacgtt ttatggccat taaagctggc tgatcgtcgc
     6241 caaacaccaa atacataatg aatatgtaca cattcgagaa agaagcgatc aaagaagcgt
     6301 cttcgggcgg agtaggagaa tgcggaggag aaggagaacg agctgatcta gtatctctcc
     6361 acaatccaat gccaactgac caactggcca tattcggagc aatttgaagc caatttccat
     6421 cgcctggcga tcgctccatt cttggctata tgtttttcac cgttacccgg ggccattttc
     6481 aaagactcgt cggcaagata agattgtgtc actcgctgtc tctcttcatt tgtcgaagaa
     6541 tgctgaggaa tttcgcgatg acgtcggcga gtattttgaa gaatgagaat aatttgtatt
     6601 tatacgaaaa tcagttagtg gaattttcta caaaaacatg ttatctatag ataattttgt
     6661 tgcaaaatat gttgactatg acaaagattg tatgtatatc tttaatgtat tctcattttc
     6721 ttatgtattt ataatggcaa tgatgatact gatgatattt taagatgatg ccagaccaaa
     6781 aggcttgaat ttctgcgtct tttgccgaac gcagtgcatg tgcaattgtt gttttttgga
     6841 atattcaatt ttcggactgt ccgctttgat ttcagtttct tggcttattc aaaaagcaaa
     6901 gtaaagccaa aaaagcgaga tggcaatacc aaatgcggca aaacggtagt ggaaggaaag
     6961 gggtgcgggg cagcggaagg aagggtgggg cggggcgtgg cgg
//
