LOCUS       sequence-340109-        5529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid containing the HDR template for the in-frame, 
            N-terminal 2xFLAG-mAID tagging of AMBRA1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5529)
  AUTHORS   Simoneschi D, Rona G, Zhou N, Jeong YT, Jiang S, Milletti G, Arbini 
            AA, O'Sullivan A, Wang AA, Nithikasem S, Keegan S, Siu Y, 
            Cianfanelli V, Maiani E, Nazio F, Cecconi F, Boccalatte F, Fenyo D, 
            Jones DR, Busino L, Pagano M
  TITLE     CRL4(AMBRA1) is a master regulator of D-type cyclins.
  JOURNAL   Nature. 2021 Apr;592(7856):789-793. doi: 10.1038/s41586-021-03445-y.
            Epub 2021 Apr 14.
  PUBMED    33854235
REFERENCE   2  (bases 1 to 5529)
  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"; doi: 
            "10.1038/s41586-021-03445-y"; journalName: "Nature"; date: 
            "2021-04"; volume: "592"; issue: "7856"; pages: "789-793"
COMMENT     Sequence Label: pUC57-2xFLAG-mAID-AMBRA1 HDR donor template
FEATURES             Location/Qualifiers
     source          1..5529
                     /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             1620..1643
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1659..1682
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1728..1931
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     primer_bind     complement(3308..3324)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3308..3324)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3321..3343)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3332..3348
                     /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(3356..3362,3363..3380,3381..3386))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3401..3422
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3539..3556)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3710..4298)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3790..3809)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4469..5260,5261..5329))
                     /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     5092..5111
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5330..5434)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5502..5520
                     /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 gacgcgtatt gggatggatc
      421 caacatcctt cttcctagga gttgcataat gaattaaata tgcagggtta aagtgtcttg
      481 atacctactg aaataattcc aaaattatac aaaaaaaaca ccaacctcaa tgtaaccata
      541 tctctatcca tggagataga tgaggcaaat ttggcaaaat attgacaatt cttgagtcta
      601 ggtggagaat agtgtatgtt tattgtacca ttctttcaac tttcctatat tttttaaaac
      661 attcatagtg gagctaaaga aacaatgtca cagtggataa cactgtatat atgtagatat
      721 tcttttgaaa taccatttta tcaggcttac atagtccatt gtatagacca caatttgttc
      781 tttaatcatt acacttgtgt gtattgtgct gctagttgcc attttagtag aattttaatt
      841 tctgatgaag ttttctcctt gtaagtcttg tggtagatga gaaaaggagc ctgatttttt
      901 tttttccttc cctttccttc tctgtcctct accctggatc tttagtggag aagagaaaga
      961 gaaaatgagt gagaaaggta ctcctaaata atcttttttt ttttgagatg gagtcttgct
     1021 ctgtcgccca ggcactggag tgcaatggca ctatctcagc tctctgcaac ttctgcgtcc
     1081 tgggttcaag caattctcct gcttcagtct gatgagtagc tgggattaca ggtgcctgcc
     1141 accacacccg gctaattttt gtattttagt agagacgggg tttcaccatg ttggccaggc
     1201 tgtctcaaac tcctgacctc aagcaatcca atctccttgg cttcccaaag ttcggggatt
     1261 acaggcgtga gccactgtgc ttggcctgat cctttcttta gctacaaggt tgagctatgg
     1321 agaatatgag agtatgaaat atttttggaa gaaaaattct gggaggaagg gtcttttctg
     1381 agaaagtgtc attaaatagg aaaagtataa tttccctttt gagctagaat tgtatttgca
     1441 attagaagct tctcgttgca gaagtcgttc tttgacatta ttctttgttc cttaagattt
     1501 ccatgaggac aacttacaag gacctgtatg gcctcatttt agctgcttca gtgtgttgga
     1561 gctgttactt gctccttcat ttcctgggcc tggtgtgaca gtggctactg agcgccatgg
     1621 actacaagga cgacgatgac aaggtaccag gatctgggga ttataaagat gatgatgata
     1681 aaggaggagg aggaagcggc ggaggcggaa gcggaggagg aggaagcaag gagaagagtg
     1741 cttgtcctaa agatccagcc aaacctccgg ccaaggcaca agttgtggga tggccaccgg
     1801 tgagatcata ccggaagaac gtgatggttt cctgccaaaa atcaagcggt ggcccggagg
     1861 cggcggcgtt cgtgaaggta tcaatggacg gagcaccgta cttgaggaaa atcgatttga
     1921 ggatgtataa aggaggagga ggaagcggcg gaggcggaag cggaggagga ggaagcatga
     1981 aggttgtccc agaaaagaat gctgtccgga tactctgggg gcgagaacgg ggtgctcggg
     2041 ccatgggagc tcagcggctt ctgcaggagc tggtagaaga taaaacccgg tggatgaaat
     2101 gggagggcaa ggtaaggaaa atggctatat ttcacatagg atttgtgctg gtgatggtag
     2161 aatgagacct tagaatgggt taacagcaga gagttgggat atatttatct tgtgtgggag
     2221 agttgacata tctgacttag ggagttgttt gtaaatttga aagaactaat gtgctagaaa
     2281 tatctttttc ctttagagct agacagtttt cctgaaggta atttaggttt atctctctca
     2341 atttgtagct tgatcattgt ctccagtttt taagcctaca ctgtgaatgc taatctagaa
     2401 tggttaatgt gcttttgaga tacggtgaac catggtctac aaatcatgta ttttaacttt
     2461 tttttttttt ttgtctccca gagagtagaa ctgccggata gtccacgctc taccttctta
     2521 ttggccttca gcccagacag gtactcagta tttatctata acttaagatt tgtgataaca
     2581 gtgctttctg tatatttggc ctggcttgat cttccaagtc agtccctaaa gtctctctga
     2641 agtactgtat gaacagcact ttggccccgt gtcggaaagg gaaagaaagt agatgagcag
     2701 tgctaacctg aaaaatcatg agaacaagca aaggaaactg agaggatcaa aacttggaaa
     2761 gtttctcaat aacaatcatt tgcctgtgtg atacagctca ccagtggtat gatggttttt
     2821 ccagtgggaa aaaatatttt ccgaatgtgg catgtgtcat ttggggacag tgtggacaag
     2881 ccaaaagctt ttaaagatca gaatatttct gtttgtagat atactgccta acatacaaag
     2941 cttagtttct ttcatgttgg gattggcagg tagcatcaat attctatgtc gtccaaagtt
     3001 gataagattg gttacccttc cacatgcttc tgaattcagt tctactcttt gattttctag
     3061 gactctctta gcctccaccc atgtgaacca taatatctat attacggagg tgaagactgg
     3121 caagtgtgtt cattccctga ttggacaccg ccgtactcca tggtgtgtca cttttcatcc
     3181 caccatctca ggccttattg cttctggctg cctagatggg gaggttagga tttgggattt
     3241 acacgtgagt atttccttaa gatacccctc gagatcccaa tggcgcgccg agcttggcgt
     3301 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3361 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3421 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3481 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3541 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3601 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3661 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3721 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3781 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3841 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3901 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3961 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4021 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4081 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4141 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4201 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4261 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4321 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4381 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4441 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4501 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4561 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4621 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4681 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4741 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4801 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4861 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4921 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4981 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5041 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5101 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5161 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5221 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5281 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5341 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5401 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5461 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5521 cctttcgtc
//
