LOCUS       sequence-234557-        4366 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4366)
  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 4366)
  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: pMK348 (BSD-P2A-mAID-mClover)
FEATURES             Location/Qualifiers
     source          1..4366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..89
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     81..98
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     82..98
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     108..127
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        108..126
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     151..167
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     152..168
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      183..192
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             189..584
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             597..653
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             660..863
                     /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(870..872,873..875,876..1586)
                     /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(915..936)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1523..1544
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1606..1622)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1606..1622)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1659..1679)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1659..1677)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1698..1714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1698..1714)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1711..1733)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1722..1738
                     /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(1746..1752,1753..1770,1771..1776))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1791..1812
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1929..1946)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2100..2688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2180..2199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2859..3650,3651..3719))
                     /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     3482..3501
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3720..3824)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3851..4307
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3938..3957)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4148..4169
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
       61 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
      121 tatagggcga attgggtacc gggccccccc tcgaggtcga cggtatcgat aagcttgata
      181 tcgccaccat ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg
      241 ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta
      301 gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag
      361 aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg
      421 cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc
      481 tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag
      541 ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctcagga agcggagcta
      601 ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctagatcta
      661 aggagaagag tgcttgtcct aaagatccag ccaaacctcc ggccaaggca caagttgtgg
      721 gatggccacc ggtgagatca taccggaaga acgtgatggt ttcctgccaa aaatcaagcg
      781 gtggcccgga ggcggcggcg ttcgtgaagg tatcaatgga cggagcaccg tacttgagga
      841 aaatcgattt gaggatgtat aaagctagca tggtgagcaa gggcgaggag ctgttcaccg
      901 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtcc
      961 gcggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca
     1021 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcgtggcct
     1081 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1141 aaggctacgt ccaggagcgc accatctctt tcaaggacga cggtacctac aagacccgcg
     1201 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1261 tcaaggagga cggcaacatc ctggggcaca agctggagta caacttcaac agccacaacg
     1321 tctatatcac ggccgacaag cagaagaacg gcatcaaggc taacttcaag atccgccaca
     1381 acgttgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1441 acggccccgt gctgctgccc gacaaccact acctgagcca tcagtccaag ctgagcaaag
     1501 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatta
     1561 cacatggcat ggacgagctg tacaagggcg ctggtgcagg cgccggatcc actagttcta
     1621 gagcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg
     1681 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1741 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1801 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1861 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1921 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1981 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2041 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2101 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2161 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2221 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2281 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2341 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2401 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2461 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2521 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2581 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2641 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2701 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2761 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2821 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2881 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2941 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3001 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3061 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3121 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3181 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3241 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3301 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3361 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3421 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3481 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3541 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3601 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3661 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3721 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3781 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3841 gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3901 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3961 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4021 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4081 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4141 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4201 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaaatg agctgattta
     4261 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc
     4321 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgc
//
