LOCUS       sequence-331497-        4806 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes BirA-FLAG with left and right homology arms for endogenous 
            tagging of Ago1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4806)
  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 4806)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_Ago1
FEATURES             Location/Qualifiers
     source          1..4806
                     /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(991..1599,1600..1602,1603..1953))
                     /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(2005..2028)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2585..2601)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2585..2601)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2598..2620)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2609..2625
                     /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(2633..2639,2640..2657,2658..2663))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2678..2699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2816..2833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2987..3575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3067..3086)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3746..4537,4538..4606))
                     /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     4369..4388
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4607..4711)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4779..4797
                     /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 ccgcgccacc actcaagtcc ccaatctaag gggggaaatc cctggtctaa atccccaccc
      481 accgccccct gaccctcacc cccgcttcag gcccagctgg gcggagctca gaggccagcc
      541 ccggcgccta ctccccagag acattagctt agcccagccc agcacggccg agcccccgcc
      601 ccccgacagg cggtagttcc aggcccatct gtgtcgcccc cctcctctcc aggacacctg
      661 gagcccgccc ccctgccacc tgccgggcct ccccataggc ctccccagac ctcacggaag
      721 gacaccgtgc attcaggccc ttctcccagg ttacttctca ccctcgggac agtatactgc
      781 ttcctaggac aaacagcccg ggtctcagat cttcacatgg ttctcagccg gaactgcata
      841 caaccccctt caaaatgggc cccccatccc ctgagagccc ctctcctcac tctgcaacca
      901 tgctctatgc cgcgagagcc tatagggctt tggaatacac actgctccca gtccccgagg
      961 gcccttacct gctcccgagg gtcccgcttc cttctcggcg ctccgcaggc tgatctcgcc
     1021 gcccatccag ggcttgatga tgccgtcctg ctccagcagc agggcgccct gcttgtcgat
     1081 gccccggctg atgccgaaga tctccttgtc gccgatgatc agcttcacgg gccggttgat
     1141 gaagttgtcc agcttctccc accggctcag gtagggggcc aggccctcct gctcgaacag
     1201 ctccagggcg gcccgcagct cccggatcag catggcggcc agggtgttcc ggtccaggtt
     1261 gatgccggcc tcctgcaggg tgatccagcc ctggttcacc acgctctcct ccacccgccg
     1321 catggccatg ttgatgccgg cgccgatcac gatctgggcg gcgtcgccgg tcttgccggt
     1381 cagctccacc aggatgccgg ccagcttccg gtcctgcagg tacaggtcgt tgggccactt
     1441 cacccgcacc ttgtcggcgc ccagcttccg cagcacctcg gccatcacga tgccgatcac
     1501 caggctcagg ccgatggcgg cggcggggcc ctgctccagc cgccagaaca tgctcaggta
     1561 caggttggcg ccgaaggggc tgaaccactt ccggccccgg ccgccccggc cggcctgctg
     1621 gtactcggcg atgcaggcgt cgccgctctt cagctcgccg atccggtcca gcaggtactg
     1681 gttggtgctg tcgatcacgg gcagcacggc cacgctgccg ccgtccagct ggcccaggat
     1741 ctgcttggcg ttcagcagct ggatgggctc gggcaggctg tagcccttgc cgggcacggt
     1801 gaacacgtcc acgccccagt cccgcagggt ctggatgtgc ttgttgatgg cggcccggct
     1861 catgcccagg gtctcgccca gctgctcgcc gctgtggaac tcgccgttgg ccagcagggc
     1921 gatcagcttc aggggcacgg tgttgtcctt catggtggcg ctagctggat ccttaattaa
     1981 tggcgcaccg tttaaactgg cggctttatc gtcatcgtct ttgtagtcca tcccatatac
     2041 ccgtgcggag gacaggcggc cgggagactg aggagtccag cgtgaggggc ctaggtacct
     2101 cgcacttgca ctgctcgggc tcccgggccg ggctcggctc ccccccgaag cccggccgct
     2161 cgcgctgtcg cccccgcagc ctccgttgcc gccgccgcgg agcctgcagc agctccccct
     2221 gctagcgccc gagtagctgc cagtgcgcag gcgcgaccga tgggcccatg gggcggacgg
     2281 gaacgagtca agcggggcgc tcgacggcaa caaaggccaa tggagagggg cgggggcgcg
     2341 gggaccgtgc agcggcctct ggcggccact cgtagccgtg cagcctggcg ggagggctcc
     2401 agtggcggag ccgcgcagtg gcgtaagagg ggaggggctg ggcgacaccc cttccccggc
     2461 tgcagcctgg ggccctgtct tttccctaaa cagatctctg gaaacctatc cacagccccg
     2521 ctatcaacac agagcctgat cctctagagt cgacctgcag gcatgcaagc ttggcgtaat
     2581 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     2641 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     2701 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     2761 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     2821 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2881 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2941 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3001 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3061 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3121 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3181 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3241 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3301 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3361 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3421 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3481 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3541 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3601 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3661 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3721 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3781 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3841 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3901 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3961 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4021 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4081 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4141 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4201 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4261 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4321 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4381 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4441 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4501 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4561 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4621 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4681 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4741 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     4801 ttcgtc
//
