LOCUS       sequence-331858-        4839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with mAID-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4839)
  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 4839)
  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: pMK437 (mAID-mCherry-P2A-BSD)
FEATURES             Location/Qualifiers
     source          1..4839
                     /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..1592
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1030..1048)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             1599..1655
                     /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             1662..2060
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    2563..2596
                     /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(2645..2665)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2645..2663)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2684..2700)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2684..2700)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2697..2719)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2708..2724
                     /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(2732..2738,2739..2756,2757..2762))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2777..2798
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2915..2932)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3086..3674)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3166..3185)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3845..4636,4637..4705))
                     /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     4468..4487
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4706..4810)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(4837..4839,1..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 tgtataaagc tagcatggtg agcaagggcg
      901 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
      961 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1021 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1081 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     1141 actacttgaa gctgtccttc cccgagggct tcaattggga gcgcgtgatg aacttcgagg
     1201 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     1261 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag tgtcgtacca
     1321 tgggctggga ggcctccact gagcggatgt accccgagga cggcgccctg aagggcgaga
     1381 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1441 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcgacatc aagttggaca
     1501 tcctttccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     1561 actccaccgg cggcatggac gagctgtaca agacgcgtgc tactaacttc agcctgctga
     1621 agcaggctgg agacgtggag gagaaccctg gacctctcga gatggccaag cctttgtctc
     1681 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg
     1741 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca
     1801 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg
     1861 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct
     1921 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca
     1981 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg
     2041 gttatgtgtg ggagggctaa tcgatgatat cagatccccg ggatgcagaa attgatgatc
     2101 tattaaacaa taaagatgtc cactaaaatg gaagtttttc ctgtcatact ttgttaagaa
     2161 gggtgagaac agagtaccta cattttgaat ggaaggattg gagctacggg ggtgggggtg
     2221 gggtgggatt agataaatgc ctgctcttta ctgaaggctc tttactattg ctttatgata
     2281 atgtttcata gttggatatc ataatttaaa caagcaaaac caaattaagg gccagctcat
     2341 tcctcccact catgatctat agatctatag atctctcgtg ggatcattgt ttttctcttg
     2401 attcccactt tgtggttcta agtactgtgg tttccaaatg tgtcagtttc atagcctgaa
     2461 gaacgagatc agcagcctct gttccacata cacttcattc tcagtattgt tttgccaagt
     2521 tctaattcca tcagaagctg gtcgagatcc ggaaccctta atataacttc gtataatgta
     2581 tgctatacga agttattagg tccctcgaag aggttcacta ggatccggta cccagctttt
     2641 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     2701 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     2761 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     2821 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2881 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2941 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3001 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3061 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3121 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3181 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3241 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3301 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3361 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3421 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3481 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3541 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3601 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3661 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3721 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3781 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3841 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3901 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3961 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4021 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4081 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4141 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4201 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4261 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4321 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4381 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4441 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4501 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4561 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4621 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4681 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4741 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4801 gggttccgcg cacatttccc cgaaaagtgc cacctgacg
//
