LOCUS       sequence-230642-        5674 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 5674)
  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 5674)
  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: pMK392 (mAID-mClover-BSD)
FEATURES             Location/Qualifiers
     source          1..5674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             681..884
                     /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(891..893,894..896,897..1610)
                     /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(936..957)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1544..1565
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1728..1849
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1765..1784)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1819..1838
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1880..1913
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1974..2473
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2009..2032)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2406..2425
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2497..2506
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2503..2901
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    3404..3437
                     /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(3486..3506)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3486..3504)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3525..3541)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3525..3541)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3538..3560)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3549..3565
                     /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(3573..3579,3580..3597,3598..3603))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3618..3639
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3756..3773)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3927..4515)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4007..4026)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4686..5477,5478..5546))
                     /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     5309..5328
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5547..5651)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctccc
      661 cgggatccgg tgcaggcgcc aaggagaaga gtgcttgtcc taaagatcca gccaaacctc
      721 cggccaaggc acaagttgtg ggatggccac cggtgagatc ataccggaag aacgtgatgg
      781 tttcctgcca aaaatcaagc ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg
      841 acggagcacc gtacttgagg aaaatcgatt tgaggatgta taaagctagc atggtgagca
      901 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      961 acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac ggcaagctga
     1021 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1081 ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1141 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct ttcaaggacg
     1201 acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1261 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1321 acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg
     1381 ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc gaccactacc
     1441 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcc
     1501 atcagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1561 tcgtgaccgc cgccgggatt acacatggca tggacgagct gtacaagtaa tctagataac
     1621 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     1681 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     1741 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1801 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtcgatc
     1861 atattcaata acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtct
     1921 gaagaggagt ttacgtccag ccaagcttag gatctcgacc tcgaaattct accgggtagg
     1981 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     2041 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     2101 gactccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     2161 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     2221 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     2281 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     2341 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     2401 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     2461 tccgggcctt tcgacctgca tccatctaga tctcgagcca ccatggccaa gcctttgtct
     2521 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     2581 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     2641 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     2701 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     2761 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     2821 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     2881 ggttatgtgt gggagggcta atcgatgata tcagatcccc gggatgcaga aattgatgat
     2941 ctattaaaca ataaagatgt ccactaaaat ggaagttttt cctgtcatac tttgttaaga
     3001 agggtgagaa cagagtacct acattttgaa tggaaggatt ggagctacgg gggtgggggt
     3061 ggggtgggat tagataaatg cctgctcttt actgaaggct ctttactatt gctttatgat
     3121 aatgtttcat agttggatat cataatttaa acaagcaaaa ccaaattaag ggccagctca
     3181 ttcctcccac tcatgatcta tagatctata gatctctcgt gggatcattg tttttctctt
     3241 gattcccact ttgtggttct aagtactgtg gtttccaaat gtgtcagttt catagcctga
     3301 agaacgagat cagcagcctc tgttccacat acacttcatt ctcagtattg ttttgccaag
     3361 ttctaattcc atcagaagct ggtcgagatc cggaaccctt aatataactt cgtataatgt
     3421 atgctatacg aagttattag gtccctcgaa gaggttcact aggatccggt acccagcttt
     3481 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     3541 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3601 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3661 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3721 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3781 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3841 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3901 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3961 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4021 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4081 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4141 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4201 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4261 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4321 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     4381 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4441 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4501 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4561 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4621 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4681 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4741 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4801 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4861 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4921 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4981 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5041 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5101 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5161 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5221 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5281 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5341 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5401 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5461 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5521 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5581 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5641 ggggttccgc gcacatttcc ccgaaaagtg ccac
//
