LOCUS       sequence-234567-        5674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5674)
  AUTHORS   Yesbolatova A, Natsume T, Hayashi KI, Kanemaki MT
  TITLE     Generation of conditional auxin-inducible degron (AID) cells and 
            tight control of degron-fused proteins using the degradation 
            inhibitor auxinole.
  JOURNAL   Methods. 2019 Apr 23. pii: S1046-2023(18)30331-1. doi: 
            10.1016/j.ymeth.2019.04.010.
  PUBMED    31026591
REFERENCE   2  (bases 1 to 5674)
  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: "Methods. 
            2019 Apr 23. pii: S1046-2023(18)30331-1. doi: 
            10.1016/j.ymeth.2019.04.010."
COMMENT     Sequence Label: pMK392 (mAID-mClover-BSD)
FEATURES             Location/Qualifiers
     source          1..5674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..96
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     88..105
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     89..105
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     115..134
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        115..133
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             170..373
                     /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(380..382,383..385,386..1099)
                     /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(425..446)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1033..1054
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1217..1338
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1254..1273)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1308..1327
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1369..1402
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1463..1962
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1498..1521)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1895..1914
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1986..1995
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1992..2390
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    2893..2926
                     /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(2975..2995)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2975..2993)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3014..3030)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3014..3030)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3027..3049)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3038..3054
                     /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(3062..3068,3069..3086,3087..3092))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3107..3128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3245..3262)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3416..4004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3496..3515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4175..4966,4967..5035))
                     /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     4798..4817
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5036..5140)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5167..5622
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5254..5273)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5464..5485
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
       61 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
      121 gactcactat agggcgaatt ggagctcccc gggatccggt gcaggcgcca aggagaagag
      181 tgcttgtcct aaagatccag ccaaacctcc ggccaaggca caagttgtgg gatggccacc
      241 ggtgagatca taccggaaga acgtgatggt ttcctgccaa aaatcaagcg gtggcccgga
      301 ggcggcggcg ttcgtgaagg tatcaatgga cggagcaccg tacttgagga aaatcgattt
      361 gaggatgtat aaagctagca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
      421 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtcc gcggcgaggg
      481 cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
      541 gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcgtggcct gcttcagccg
      601 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
      661 ccaggagcgc accatctctt tcaaggacga cggtacctac aagacccgcg ccgaggtgaa
      721 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
      781 cggcaacatc ctggggcaca agctggagta caacttcaac agccacaacg tctatatcac
      841 ggccgacaag cagaagaacg gcatcaaggc taacttcaag atccgccaca acgttgagga
      901 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
      961 gctgctgccc gacaaccact acctgagcca tcagtccaag ctgagcaaag accccaacga
     1021 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatta cacatggcat
     1081 ggacgagctg tacaagtaat ctagataact gatcataatc agccatacca catttgtaga
     1141 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     1201 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1261 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1321 actcatcaat gtatcttaac gcgtcgatca tattcaataa cccttaatat aacttcgtat
     1381 aatgtatgct atacgaagtt attaggtctg aagaggagtt tacgtccagc caagcttagg
     1441 atctcgacct cgaaattcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca
     1501 tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg gcctcgcaca
     1561 cattccacat ccaccggtag gcgccaaccg actccgttct ttggtggccc cttcgcgcca
     1621 ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg
     1681 acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag caccgctgag
     1741 caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc cttcgctttc
     1801 tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc
     1861 ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg
     1921 tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcat ccatctagat
     1981 ctcgagccac catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
     2041 cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc gcagctctct
     2101 ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg ggaccttgtg
     2161 cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg acttgtatcg
     2221 tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgc cgacaggtgc
     2281 ttctcgatct gcatcctggg atcaaagcca tagtgaagga cagtgatgga cagccgacgg
     2341 cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa tcgatgatat
     2401 cagatccccg ggatgcagaa attgatgatc tattaaacaa taaagatgtc cactaaaatg
     2461 gaagtttttc ctgtcatact ttgttaagaa gggtgagaac agagtaccta cattttgaat
     2521 ggaaggattg gagctacggg ggtgggggtg gggtgggatt agataaatgc ctgctcttta
     2581 ctgaaggctc tttactattg ctttatgata atgtttcata gttggatatc ataatttaaa
     2641 caagcaaaac caaattaagg gccagctcat tcctcccact catgatctat agatctatag
     2701 atctctcgtg ggatcattgt ttttctcttg attcccactt tgtggttcta agtactgtgg
     2761 tttccaaatg tgtcagtttc atagcctgaa gaacgagatc agcagcctct gttccacata
     2821 cacttcattc tcagtattgt tttgccaagt tctaattcca tcagaagctg gtcgagatcc
     2881 ggaaccctta atataacttc gtataatgta tgctatacga agttattagg tccctcgaag
     2941 aggttcacta ggatccggta cccagctttt gttcccttta gtgagggtta attgcgcgct
     3001 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3061 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     3121 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3181 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3241 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3301 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3361 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3421 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3481 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3541 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3601 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3661 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3721 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3781 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3841 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3901 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3961 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4021 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4081 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4141 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4201 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4261 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4321 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4381 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4441 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4501 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4561 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4621 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4681 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4741 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4801 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4861 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4921 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4981 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5041 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5101 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5161 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5221 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5281 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5341 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5401 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5461 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     5521 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5581 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttccattcgc cattcaggct
     5641 gcgcaactgt tgggaagggc gatcggtgcg ggcc
//
