LOCUS       sequence-367851-        4141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal tag cassette with BbsI restriction sites for CRISPR donor
            plasmid. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4141)
  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 4141)
  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_Ntag_BbsI-Mammalian
FEATURES             Location/Qualifiers
     source          1..4141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..22
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(30..387)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      44..179
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        196..387
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     344..364
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             435..500
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             537..626
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(721..738)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(721..737)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(730..752)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(946..965)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1065..1087
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1125..1143)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1211..1315
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1316..1384,1385..2176)
                     /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(1534..1553)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2347..2935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2836..2855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3089..3106
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3223..3244
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3259..3264,3265..3282,3283..3289)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3297..3313
                     /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     3302..3324
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3321..3337
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3321..3337
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3429..3563
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3453..3472)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3507..3526
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(3564..4141,1..22))
                     /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(3647..3667)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 ccgtgggctt gtactcggtc attttgattt ttgcaaaagc ctaggcctcc aaaaaagcct
       61 cctcactact tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa
      121 aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg
      181 ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt
      241 ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg
      301 ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca
      361 ttccacagct ggttctttcc gcctcagcta attttaatca accgatttct ctctcttccg
      421 caggaatcaa aatggactac aaagaccatg acggtgatta taaagatcat gacatcgatt
      481 acaaggatga cgatgacaag ggtggatctg gaggttccgg cggctcaggg ggtagttacc
      541 catacgatgt tcctgactat gcgggctatc cctatgacgt cccggactat gcaggatcct
      601 atccatatga cgttccagat tacgcttccg gtggctctgg ctcttcaggc tcatattatt
      661 atgtcttctg catgcctgca ggtcgactct agaggatccc cgggtaccga gctcgaattc
      721 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
      781 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
      841 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
      901 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
      961 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     1021 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     1081 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     1141 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1201 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1261 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1321 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1381 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1441 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1501 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1561 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1621 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1681 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     1741 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     1801 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     1861 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     1921 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     1981 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2041 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2101 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2161 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2221 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2281 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2341 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2401 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2461 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2521 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2581 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2641 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2701 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2761 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     2821 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2881 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     2941 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3001 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3061 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3121 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     3181 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     3241 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     3301 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca gaagacagaa
     3361 actcagtttc cattgtgaag ttttaagtga tcgcgagtgc caaaaagtaa caaaacaccc
     3421 aggtaagtga tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     3481 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     3541 ttataagctg caataaacaa gttctaggca ccgggcttgc gggtcatgca ccaggtgcgc
     3601 ggtccttcgg gcacctcgac gtcggcggtg acggtgaagc cgagccgctc gtagaagggg
     3661 aggttgcggg gcgcggaggt ctccaggaag gcgggcaccc cggcgcgctc ggccgcctcc
     3721 actccgggga gcacgacggc gctgcccaga cccttgccct ggtggtcggg cgagacgccg
     3781 acggtggcca ggaaccacgc gggctccttg ggccggtgcg gcgccaggag gccttccatc
     3841 tgttgctgcg cggccagccg ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg
     3901 aacaccgccc ccgcttcgac gctctccggc gtggtccaga ccgccaccgc ggcgccgtcg
     3961 tccgcgaccc acaccttgcc gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc
     4021 tcggtgaccc gctcgatgtg gcggtccggg tcgacggtgt ggcgcgtggc ggggtagtcg
     4081 gcgaacgcgg cggcgagggt gcgtacggcc cgggggacgt cgtcgcgggt ggcgaggcgc
     4141 a
//
