LOCUS       sequence-293810-        6646 bp    DNA     circular SYN 14-APR-2025
DEFINITION  TOP2A tagging with mAID-Clover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6646)
  AUTHORS   Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano 
            R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, 
            Hayashi KI, Kanemaki MT
  TITLE     The auxin-inducible degron 2 technology provides sharp degradation 
            control in yeast, mammalian cells, and mice.
  JOURNAL   Nat Commun. 2020 Nov 11;11(1):5701. doi: 10.1038/s41467-020-19532-z.
  PUBMED    33177522
REFERENCE   2  (bases 1 to 6646)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2020-11-11"; volume: "11(1):5701. doi"; pages: " 
            10.1038/s41467-020-19532-z"
COMMENT     Sequence Label: pMK322 (TOP2A-mAC (Hygro))
FEATURES             Location/Qualifiers
     source          1..6646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2504..2707
                     /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"
     CDS             join(2714..2716,2717..2719,2720..3433)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2759..2780)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3367..3388
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3551..3672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3588..3607)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3642..3661
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    3703..3736
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3797..4296
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3832..3855)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4229..4248
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4357..5376
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             4357..5376
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     protein_bind    5939..5972
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6252..6269)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6252..6268)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6261..6283)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6477..6496)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6596..6618
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctc ggcgcgccat tgggatggaa cgaaaactcc ttgaatatag
     2281 tgtcttgata cattttttta cattacaatt atgttgtcag atttacaatg tgcaagttac
     2341 ctgggctttt ctcttttaga aatccaaggg ggagagtgat gacttccata tggactttga
     2401 ctcagctgtg gctcctcggg caaaatctgt acgggcaaag aaacccatta aatacctgga
     2461 agagtcagat gaagatgatc tgtttggatc cggtgcaggc gccaaggaga agagtgcttg
     2521 tcctaaagat ccagccaaac ctccggccaa ggcacaagtt gtgggatggc caccggtgag
     2581 atcataccgg aagaacgtga tggtttcctg ccaaaaatca agcggtggcc cggaggcggc
     2641 ggcgttcgtg aaggtatcaa tggacggagc accgtacttg aggaaaatcg atttgaggat
     2701 gtataaagct agcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2761 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtccgcggcg agggcgaggg
     2821 cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     2881 gccctggccc accctcgtga ccaccttcgg ctacggcgtg gcctgcttca gccgctaccc
     2941 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3001 gcgcaccatc tctttcaagg acgacggtac ctacaagacc cgcgccgagg tgaagttcga
     3061 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3121 catcctgggg cacaagctgg agtacaactt caacagccac aacgtctata tcacggccga
     3181 caagcagaag aacggcatca aggctaactt caagatccgc cacaacgttg aggacggcag
     3241 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     3301 gcccgacaac cactacctga gccatcagtc caagctgagc aaagacccca acgagaagcg
     3361 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg attacacatg gcatggacga
     3421 gctgtacaag taatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     3481 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     3541 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3601 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3661 caatgtatct taacgcgtcg atcatattca ataaccctta atataacttc gtataatgta
     3721 tgctatacga agttattagg tctgaagagg agtttacgtc cagccaagct taggatctcg
     3781 acctcgaaat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
     3841 ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc
     3901 acatccaccg gtaggcgcca accgactccg ttctttggtg gccccttcgc gccaccttct
     3961 actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga
     4021 caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg
     4081 aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct
     4141 cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg
     4201 ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc
     4261 gcgctgttct cctcttcctc atctccgggc ctttcgacct gcatccatct agatctcgag
     4321 cagctgaagc ttaccgctag catggataga tccggaaagc ctgaactcac cgcgacgtct
     4381 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     4441 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     4501 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     4561 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     4621 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     4681 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     4741 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     4801 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     4861 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     4921 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     4981 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     5041 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     5101 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     5161 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     5221 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     5281 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     5341 cgacgcccca gcactcgtcc gagggcaaag gaataggcta gccgcccgcc ccacgacccg
     5401 cagcgcccga ccgaaaggag cgcacgaccc catgcatcga tgatatcaga tccccgggat
     5461 gcagaaattg atgatctatt aaacaataaa gatgtccact aaaatggaag tttttcctgt
     5521 catactttgt taagaagggt gagaacagag tacctacatt ttgaatggaa ggattggagc
     5581 tacgggggtg ggggtggggt gggattagat aaatgcctgc tctttactga aggctcttta
     5641 ctattgcttt atgataatgt ttcatagttg gatatcataa tttaaacaag caaaaccaaa
     5701 ttaagggcca gctcattcct cccactcatg atctatagat ctatagatct ctcgtgggat
     5761 cattgttttt ctcttgattc ccactttgtg gttctaagta ctgtggtttc caaatgtgtc
     5821 agtttcatag cctgaagaac gagatcagca gcctctgttc cacatacact tcattctcag
     5881 tattgttttg ccaagttcta attccatcag aagctggtcg agatccggaa cccttaatat
     5941 aacttcgtat aatgtatgct atacgaagtt attaggtccc tcgaagaggt tcactaggat
     6001 ccagagtcag atgaagatga tctgttttaa aatgtgaggc gattatttta agtaattatc
     6061 ttaccaagcc caagactggt tttaaagtta cctgaagctc ttaacttcct cccctctgaa
     6121 tttagtttgg ggaaggtgtt tttagtacaa gacatcaaag tgaagtaaag cccaagtgtt
     6181 ctttagcttt ttataatact gtctaaatag tgaccatctc atgggcgttc catcccaata
     6241 cgcgtcaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     6301 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     6361 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     6421 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     6481 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     6541 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     6601 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcga
//
