LOCUS       sequence-367468-        3507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty donor plasmid for C-terminal tag of 3xFlag-3xHA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3507)
  AUTHORS   Meng Q, Stoyko D, Andrews CM, Konstantinidou P, Genzor P, O T, 
            Elchert AR, Benner L, Sobti S, Katz EY, Haase AD
  TITLE     Functional editing of endogenous genes through rapid selection of 
            cell pools (Rapid generation of endogenously tagged genes in 
            Drosophila ovarian somatic sheath cells).
  JOURNAL   Nucleic Acids Res. 2022 May 27. pii: 6594084. doi: 
            10.1093/nar/gkac448.
  PUBMED    35639929
REFERENCE   2  (bases 1 to 3507)
  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. 2022 May 27. pii: 6594084. doi: 10.1093/nar/gkac448."
COMMENT     Sequence Label: PUC19_C_tag
FEATURES             Location/Qualifiers
     source          1..3507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..52)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(35..51)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(44..66)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(260..279)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     379..401
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(439..457)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        525..629
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(630..698,699..1490)
                     /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     complement(848..867)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1661..2249
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2150..2169
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2403..2420
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2537..2558
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2573..2578,2579..2596,2597..2603)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2611..2627
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2616..2638
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2635..2651
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2635..2651
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2698..2763
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2782..2808
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2818..2874
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2875..3474
                     /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(2875..2894)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3371..3391
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 ctctagagga tccccgggta ccgagctcga attcactggc cgtcgtttta caacgtcgtg
       61 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
      121 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      181 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
      241 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
      301 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
      361 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
      421 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
      481 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      541 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
      601 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
      661 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
      721 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
      781 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
      841 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
      901 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
      961 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1021 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1081 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1141 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     1201 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     1261 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     1321 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     1381 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     1441 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     1501 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     1561 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1621 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1681 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1741 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1801 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     1861 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     1921 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     1981 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2041 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2101 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2161 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2221 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2281 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     2341 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     2401 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     2461 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2521 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     2581 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     2641 acagctatga ccatgattac gccagaagac agaaacacca gcggaggagg ttccggtgac
     2701 tacaaggacc acgacggcga ttataaggat cacgacatcg actacaaaga cgacgatgac
     2761 aaggctggcg ccagcagcgc ctacccatac gacgtaccag attacgctgc tggatctgcc
     2821 acgaacttct ctctgttaaa gcaagcagga gacgtggaag aaaaccccgg tcctatgacc
     2881 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     2941 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     3001 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     3061 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     3121 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     3181 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     3241 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     3301 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     3361 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     3421 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctagtattat
     3481 tatgtcttct gcatgcctgc aggtcga
//
