LOCUS       sequence-242002-        5665 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 5665)
  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 5665)
  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: pMK393 (mAID-mCherry-BSD)
FEATURES             Location/Qualifiers
     source          1..5665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     144..166
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     158..175
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     159..175
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     185..204
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        185..203
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             240..443
                     /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             450..1160
                     /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(595..613)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    1278..1399
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1315..1334)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1369..1388
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1430..1463
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1524..2023
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1559..1582)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1956..1975
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2047..2056
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2053..2451
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    2954..2987
                     /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(3036..3056)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3036..3054)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3075..3091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3075..3091)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3088..3110)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3099..3115
                     /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(3123..3129,3130..3147,3148..3153))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3168..3189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3306..3323)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3477..4065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3557..3576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4236..5027,5028..5096))
                     /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     4859..4878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5097..5201)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5228..5656
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5315..5334)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5525..5546
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tattaacgct tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc
       61 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      121 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg
      181 cgcgtaatac gactcactat agggcgaatt ggagctcccc gggatccggt gcaggcgcca
      241 aggagaagag tgcttgtcct aaagatccag ccaaacctcc ggccaaggca caagttgtgg
      301 gatggccacc ggtgagatca taccggaaga acgtgatggt ttcctgccaa aaatcaagcg
      361 gtggcccgga ggcggcggcg ttcgtgaagg tatcaatgga cggagcaccg tacttgagga
      421 aaatcgattt gaggatgtat aaagctagca tggtgagcaa gggcgaggag gataacatgg
      481 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
      541 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
      601 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
      661 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
      721 ccttccccga gggcttcaat tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
      781 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
      841 gcaccaactt cccctccgac ggccccgtaa tgcagtgtcg taccatgggc tgggaggcct
      901 ccactgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
      961 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     1021 ccgtgcagct gcccggcgcc tacaacgtcg acatcaagtt ggacatcctt tcccacaacg
     1081 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     1141 tggacgagct gtacaagtaa tctagataac tgatcataat cagccatacc acatttgtag
     1201 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     1261 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     1321 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     1381 aactcatcaa tgtatcttaa cgcgtcgatc atattcaata acccttaata taacttcgta
     1441 taatgtatgc tatacgaagt tattaggtct gaagaggagt ttacgtccag ccaagcttag
     1501 gatctcgacc tcgaaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     1561 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     1621 acattccaca tccaccggta ggcgccaacc gactccgttc tttggtggcc ccttcgcgcc
     1681 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     1741 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     1801 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     1861 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     1921 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     1981 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca tccatctaga
     2041 tctcgagcca ccatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca
     2101 acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc
     2161 tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt
     2221 gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc
     2281 gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg
     2341 cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg
     2401 gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta atcgatgata
     2461 tcagatcccc gggatgcaga aattgatgat ctattaaaca ataaagatgt ccactaaaat
     2521 ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct acattttgaa
     2581 tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg cctgctcttt
     2641 actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat cataatttaa
     2701 acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta tagatctata
     2761 gatctctcgt gggatcattg tttttctctt gattcccact ttgtggttct aagtactgtg
     2821 gtttccaaat gtgtcagttt catagcctga agaacgagat cagcagcctc tgttccacat
     2881 acacttcatt ctcagtattg ttttgccaag ttctaattcc atcagaagct ggtcgagatc
     2941 cggaaccctt aatataactt cgtataatgt atgctatacg aagttattag gtccctcgaa
     3001 gaggttcact aggatccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc
     3061 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3121 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3181 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3241 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3301 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3361 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3421 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3481 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3541 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3601 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3661 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3721 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3781 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3841 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3901 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3961 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4021 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4081 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4141 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4201 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4261 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4321 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4381 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4441 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4501 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4561 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4621 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4681 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4741 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4801 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4861 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4921 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4981 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5041 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5101 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5161 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5221 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     5281 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5341 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5401 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     5461 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5521 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5581 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5641 aaatttaacg cgaattttaa caaaa
//
