LOCUS       sequence-337716-        4793 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes BirA-FLAG with left and right homology arms for endogenous 
            tagging of Ago2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4793)
  AUTHORS   Muller M, Fah T, Schaefer M, Hermes V, Luitz J, Stalder P, Arora R, 
            Ngondo RP, Ciaudo C
  TITLE     AGO1 regulates pericentromeric regions in mouse embryonic stem cells
  JOURNAL   Life Sci Alliance. 2022 Mar 2;5(6). pii: 5/6/e202101277. doi: 
            10.26508/lsa.202101277. Print 2022 Jun.
  PUBMED    35236760
REFERENCE   2  (bases 1 to 4793)
  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: "Life Sci 
            Alliance."; date: "2022-03-2"; volume: "5(6). pii: 5/6/e202101277. 
            doi"; pages: " 10.26508/lsa.202101277. Print 2022 Jun"
COMMENT     Sequence Label: pUC19_HR-BirA-FLAG_Ago2
FEATURES             Location/Qualifiers
     source          1..4793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(968..1576,1577..1579,1580..1930))
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             complement(1982..2005)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2005..2014
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2572..2588)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2572..2588)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2585..2607)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2596..2612
                     /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)."
     promoter        complement(join(2620..2626,2627..2644,2645..2650))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2665..2686
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2803..2820)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2974..3562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3054..3073)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3733..4524,4525..4593))
                     /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     4356..4375
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4594..4698)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4766..4784
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat
      421 cgagtaccgc cctccgctcg gctccaggag cggggtgggg aaatgcgtcc gtgtttttaa
      481 gttttctgga acttttccag agaaacgggc acgctggtgg gaaaggggtg ccacggcggc
      541 cacccgctcc gctaggaaat cgcagggcga ttagcacttg aatggggagg gcggctgccc
      601 ggcgtgaaac tgcgtccaag cgaccgatcg cggtcccggg gccccgtggt tcggttcccg
      661 acccgtccag ctccccgccc caggcccgcc gtgctgggcc ccacagctat gcgctcggga
      721 acggcgggag gagcgctgcg ggaggacgga gacccgttgc aggccgagcc tgggcccccg
      781 cacacgtccc ggtggcgctg ctcgggtgca ctgggcctcc cggccccgag gccctgcccg
      841 gccgccccga ggctacgccg cggccccaca ggccccgccg ccctcgcctg ccccgcccgc
      901 caaccgccaa ccgccgcggc acgagtgccg ggcgccgcgc ggactcaccg gggccggctc
      961 ccgagtactt ctcggcgctc cgcaggctga tctcgccgcc catccagggc ttgatgatgc
     1021 cgtcctgctc cagcagcagg gcgccctgct tgtcgatgcc ccggctgatg ccgaagatct
     1081 ccttgtcgcc gatgatcagc ttcacgggcc ggttgatgaa gttgtccagc ttctcccacc
     1141 ggctcaggta gggggccagg ccctcctgct cgaacagctc cagggcggcc cgcagctccc
     1201 ggatcagcat ggcggccagg gtgttccggt ccaggttgat gccggcctcc tgcagggtga
     1261 tccagccctg gttcaccacg ctctcctcca cccgccgcat ggccatgttg atgccggcgc
     1321 cgatcacgat ctgggcggcg tcgccggtct tgccggtcag ctccaccagg atgccggcca
     1381 gcttccggtc ctgcaggtac aggtcgttgg gccacttcac ccgcaccttg tcggcgccca
     1441 gcttccgcag cacctcggcc atcacgatgc cgatcaccag gctcaggccg atggcggcgg
     1501 cggggccctg ctccagccgc cagaacatgc tcaggtacag gttggcgccg aaggggctga
     1561 accacttccg gccccggccg ccccggccgg cctgctggta ctcggcgatg caggcgtcgc
     1621 cgctcttcag ctcgccgatc cggtccagca ggtactggtt ggtgctgtcg atcacgggca
     1681 gcacggccac gctgccgccg tccagctggc ccaggatctg cttggcgttc agcagctgga
     1741 tgggctcggg caggctgtag cccttgccgg gcacggtgaa cacgtccacg ccccagtccc
     1801 gcagggtctg gatgtgcttg ttgatggcgg cccggctcat gcccagggtc tcgcccagct
     1861 gctcgccgct gtggaactcg ccgttggcca gcagggcgat cagcttcagg ggcacggtgt
     1921 tgtccttcat ggtggcgcta gctggatcct taattaatgg cgcaccgttt aaactggcgg
     1981 ctttatcgtc atcgtctttg tagtccatgg tggcgttgcc gagggccgcg gccgagggcc
     2041 ggccgcgcgc tccacgggcc ccgacgccgc gagccgcgag gagccgccgg ccgctcgatc
     2101 cgccccggcg cggccgcctc gcagaacagg tttacccgac gcggccgggg cggcgggcgg
     2161 aggcggcggg agcggagggg ggggggagcg ggaggaggga ggggaggagg aggggcgaga
     2221 gggacgacga gggaggggac ggcggggagg gagggaagga ggggacagcg gggagggagg
     2281 aggcggggag gagggaaggt ggcagggaga gaggggggca ggggaggagg ctggaggcgg
     2341 cgcgcgccgg cgggatgggg ggggggggac tcgctgtggc ggccccccgc gtgcttgccc
     2401 tgaatgaggg accccagaag cgacccaggg cacgcgagcc gagaccttcc ctgcggccga
     2461 gcgaggcgac gtctggtttc cgggcgcggc cagcccggca ctgcgaccag agtcggagtc
     2521 ctccgatcct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct
     2581 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2641 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2701 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2761 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2821 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2881 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2941 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3001 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3061 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3121 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3181 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3241 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3301 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3361 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3421 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3481 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3541 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3601 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3661 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3721 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3781 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3841 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3901 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3961 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4021 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4081 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4141 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4201 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4261 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4321 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4381 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4441 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4501 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4561 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4621 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4681 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     4741 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtc
//
