LOCUS       sequence-331857-        5535 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 5535)
  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 5535)
  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: pMK436 (mAID-mCherry-P2A-Hygro)
FEATURES             Location/Qualifiers
     source          1..5535
                     /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             1677..2696
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1677..2696
                     /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    3259..3292
                     /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(3341..3361)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3341..3359)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3380..3396)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3380..3396)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3393..3415)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3404..3420
                     /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(3428..3434,3435..3452,3453..3458))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3473..3494
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3611..3628)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3782..4370)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3862..3881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4541..5332,5333..5401))
                     /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     5164..5183
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5402..5506)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5533..5535,1..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 gatggataga tccggaaagc
     1681 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     1741 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     1801 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     1861 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     1921 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     1981 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     2041 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     2101 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     2161 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     2221 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     2281 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     2341 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     2401 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     2461 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     2521 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     2581 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     2641 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataggcta
     2701 gccgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcga
     2761 tgatatcaga tccccgggat gcagaaattg atgatctatt aaacaataaa gatgtccact
     2821 aaaatggaag tttttcctgt catactttgt taagaagggt gagaacagag tacctacatt
     2881 ttgaatggaa ggattggagc tacgggggtg ggggtggggt gggattagat aaatgcctgc
     2941 tctttactga aggctcttta ctattgcttt atgataatgt ttcatagttg gatatcataa
     3001 tttaaacaag caaaaccaaa ttaagggcca gctcattcct cccactcatg atctatagat
     3061 ctatagatct ctcgtgggat cattgttttt ctcttgattc ccactttgtg gttctaagta
     3121 ctgtggtttc caaatgtgtc agtttcatag cctgaagaac gagatcagca gcctctgttc
     3181 cacatacact tcattctcag tattgttttg ccaagttcta attccatcag aagctggtcg
     3241 agatccggaa cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtccc
     3301 tcgaagaggt tcactaggat ccggtaccca gcttttgttc cctttagtga gggttaattg
     3361 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3421 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3481 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3541 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3601 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3661 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3721 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3781 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3841 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3901 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3961 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4021 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4081 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4141 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4201 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4261 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4321 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4381 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4441 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4501 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4561 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4621 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4681 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4741 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4801 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4861 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4921 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4981 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5041 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5101 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5161 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5221 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5281 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5341 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5401 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5461 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5521 aagtgccacc tgacg
//
