LOCUS       sequence-296216-        7527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SMC2 tagging with mAID-Clover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7527)
  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 7527)
  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: "Nat 
            Commun."; date: "2020-11-11"; volume: "11(1):5701. doi"; pages: " 
            10.1038/s41467-020-19532-z"
COMMENT     Sequence Label: SMC2-mAC donor (Hygro)
FEATURES             Location/Qualifiers
     source          1..7527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     677..693
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     677..693
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1260..1463
                     /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(1470..1472,1473..1475,1476..2189)
                     /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(1515..1536)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2123..2144
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2307..2428
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2344..2363)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2398..2417
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2459..2492
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2553..3052
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2588..2611)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2985..3004
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3113..4132
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             3113..4132
                     /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    4695..4728
                     /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(5293..5309)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5294..5310)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(5339..5359)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5339..5357)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5378..5394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5378..5394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5391..5413)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5402..5418
                     /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(5426..5432,5433..5450,5451..5456))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5471..5492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5609..5626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5780..6368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5860..5879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6539..7330,7331..7399))
                     /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     7162..7181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7400..7504)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgatgtttg
      721 ggctagtggt gtgctatagg aacttgaaac cttctttgat attaaaatgt cacaagaaaa
      781 atatctcact agagcagctt tagttagcaa caaaactgaa tgccagctac aaaagatagc
      841 aacaagcagt caaccaccag gaaatagtaa aattgggtaa tgaagtaccc aaagtaaaat
      901 taagagactt ctaaaacaat atggacagct agcattcatt taggtgcctc atataactat
      961 ttaaagaaat aaaattatct ccaaaccatt cttatcacca gtataaagga gtaaacttca
     1021 agtatatcac aaaaagtttt tttatacttt tttctttttt ccttaagttc attgtggtgt
     1081 cactaaaaga aggtatgttc aacaatgcaa acgttctttt caaaaccaag tttgtggatg
     1141 gtgtttctac agtagccaga tttactcaat gtcaaaatgg aaagatttca aaggaagcaa
     1201 aatccaaggc aaaaccaccc aaaggagcac acgtagaagt tggatccggt gcaggcgcca
     1261 aggagaagag tgcttgtcct aaagatccag ccaaacctcc ggccaaggca caagttgtgg
     1321 gatggccacc ggtgagatca taccggaaga acgtgatggt ttcctgccaa aaatcaagcg
     1381 gtggcccgga ggcggcggcg ttcgtgaagg tatcaatgga cggagcaccg tacttgagga
     1441 aaatcgattt gaggatgtat aaagctagca tggtgagcaa gggcgaggag ctgttcaccg
     1501 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtcc
     1561 gcggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca
     1621 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcgtggcct
     1681 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1741 aaggctacgt ccaggagcgc accatctctt tcaaggacga cggtacctac aagacccgcg
     1801 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1861 tcaaggagga cggcaacatc ctggggcaca agctggagta caacttcaac agccacaacg
     1921 tctatatcac ggccgacaag cagaagaacg gcatcaaggc taacttcaag atccgccaca
     1981 acgttgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2041 acggccccgt gctgctgccc gacaaccact acctgagcca tcagtccaag ctgagcaaag
     2101 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatta
     2161 cacatggcat ggacgagctg tacaagtaat ctagataact gatcataatc agccatacca
     2221 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     2281 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     2341 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2401 gtttgtccaa actcatcaat gtatcttaac gcgtcgatca tattcaataa cccttaatat
     2461 aacttcgtat aatgtatgct atacgaagtt attaggtctg aagaggagtt tacgtccagc
     2521 caagcttagg atctcgacct cgaaattcta ccgggtaggg gaggcgcttt tcccaaggca
     2581 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
     2641 gcctcgcaca cattccacat ccaccggtag gcgccaaccg actccgttct ttggtggccc
     2701 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
     2761 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
     2821 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
     2881 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
     2941 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
     3001 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcat
     3061 ccatctagat ctcgagcagc tgaagcttac cgctagcatg gatagatccg gaaagcctga
     3121 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
     3181 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     3241 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     3301 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
     3361 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     3421 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc
     3481 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     3541 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     3601 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     3661 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     3721 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     3781 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     3841 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     3901 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     3961 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     4021 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     4081 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat aggctagccg
     4141 cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcgatgat
     4201 atcagatccc cgggatgcag aaattgatga tctattaaac aataaagatg tccactaaaa
     4261 tggaagtttt tcctgtcata ctttgttaag aagggtgaga acagagtacc tacattttga
     4321 atggaaggat tggagctacg ggggtggggg tggggtggga ttagataaat gcctgctctt
     4381 tactgaaggc tctttactat tgctttatga taatgtttca tagttggata tcataattta
     4441 aacaagcaaa accaaattaa gggccagctc attcctccca ctcatgatct atagatctat
     4501 agatctctcg tgggatcatt gtttttctct tgattcccac tttgtggttc taagtactgt
     4561 ggtttccaaa tgtgtcagtt tcatagcctg aagaacgaga tcagcagcct ctgttccaca
     4621 tacacttcat tctcagtatt gttttgccaa gttctaattc catcagaagc tggtcgagat
     4681 ccggaaccct taatataact tcgtataatg tatgctatac gaagttatta ggtccctcga
     4741 agaggttcac taggatccac tacaaagtta tttcttcatc ttgacctgtt tttttaaatg
     4801 taaactttta aggacttgag ataactaatt tgtttatata caaaaattaa tgttactgtg
     4861 ttacttaacc catgttttct ctttatataa tcacttatcg cttacaaatg agcatatatt
     4921 cctcatctct taactagtct aattatggtc caattattgt ggttgtgatt ttatgcatat
     4981 catcaaatgt ttttttctta tgcgggtctt ttatatatta gggatcctga gatacccgat
     5041 tctatatgta aaagctaata tacaaaaaag cagattaaat tacatgataa atgtagctga
     5101 tcatcagtgt ttaattgatt taattcctaa ggcaatatta atgtgttttc taaagtcatt
     5161 caggtaagga agaattataa atcaggtaac tggaccaaca aagggaacac atataaagct
     5221 attatgcatg catggagccg tttgaggcta gttttttaag gccacaactc cagacccctg
     5281 aatcaagctt atcgataccg tcgacctcga gggggggccc ggtacccagc ttttgttccc
     5341 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5401 attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag tgtaaagcct
     5461 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5521 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5581 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5941 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6181 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6301 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6361 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6541 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6601 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6661 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6721 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6781 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6841 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6901 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6961 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7021 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7081 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7141 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7201 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7261 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7321 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7381 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7441 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7501 cgcgcacatt tccccgaaaa gtgccac
//
