LOCUS       sequence-331853-        4125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with mAID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4125)
  AUTHORS   Saito Y, Kanemaki MT
  TITLE     Targeted Protein Depletion Using the Auxin-Inducible Degron 2 (AID2)
            System.
  JOURNAL   Curr Protoc. 2021 Aug;1(8):e219. doi: 10.1002/cpz1.219.
  PUBMED    34370399
REFERENCE   2  (bases 1 to 4125)
  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: "Curr 
            Protoc."; date: "2021-08"; volume: "1(8):e219. doi"; pages: " 
            10.1002/cpz1.219"
COMMENT     Sequence Label: pMK433 (mAID-P2A-BSD)
FEATURES             Location/Qualifiers
     source          1..4125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(85..104)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     295..316
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     579..601
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     593..610
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     594..610
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     620..639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        620..638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             675..878
                     /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             885..941
                     /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             948..1346
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    1849..1882
                     /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(1931..1951)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1931..1949)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1970..1986)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1970..1986)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1983..2005)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1994..2010
                     /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(2018..2024,2025..2042,2043..2048))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2063..2084
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2201..2218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2372..2960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2452..2471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3131..3922,3923..3991))
                     /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     3754..3773
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3992..4096)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(4123..4125,1..453)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
       61 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
      121 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
      181 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
      241 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
      301 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
      361 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
      421 cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc tgcgcaactg
      481 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
      541 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
      601 gacggccagt gagcgcgcgt aatacgactc actatagggc gaattggagc tccccgggat
      661 ccggtgcagg cgccaaggag aagagtgctt gtcctaaaga tccagccaaa cctccggcca
      721 aggcacaagt tgtgggatgg ccaccggtga gatcataccg gaagaacgtg atggtttcct
      781 gccaaaaatc aagcggtggc ccggaggcgg cggcgttcgt gaaggtatca atggacggag
      841 caccgtactt gaggaaaatc gatttgagga tgtataaaac gcgtgctact aacttcagcc
      901 tgctgaagca ggctggagac gtggaggaga accctggacc tctcgagatg gccaagcctt
      961 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
     1021 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
     1081 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
     1141 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
     1201 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     1261 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     1321 cctctggtta tgtgtgggag ggctaatcga tgatatcaga tccccgggat gcagaaattg
     1381 atgatctatt aaacaataaa gatgtccact aaaatggaag tttttcctgt catactttgt
     1441 taagaagggt gagaacagag tacctacatt ttgaatggaa ggattggagc tacgggggtg
     1501 ggggtggggt gggattagat aaatgcctgc tctttactga aggctcttta ctattgcttt
     1561 atgataatgt ttcatagttg gatatcataa tttaaacaag caaaaccaaa ttaagggcca
     1621 gctcattcct cccactcatg atctatagat ctatagatct ctcgtgggat cattgttttt
     1681 ctcttgattc ccactttgtg gttctaagta ctgtggtttc caaatgtgtc agtttcatag
     1741 cctgaagaac gagatcagca gcctctgttc cacatacact tcattctcag tattgttttg
     1801 ccaagttcta attccatcag aagctggtcg agatccggaa cccttaatat aacttcgtat
     1861 aatgtatgct atacgaagtt attaggtccc tcgaagaggt tcactaggat ccggtaccca
     1921 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     1981 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     2041 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2101 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2161 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2221 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2281 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2341 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2401 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2461 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2521 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2581 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2641 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2701 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2761 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2821 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2881 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2941 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3001 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3061 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3121 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3181 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3241 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3301 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3361 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3421 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3481 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3541 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3601 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3661 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3721 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3781 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3841 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3901 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3961 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4021 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4081 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacg
//
