LOCUS       sequence-234558-        4357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4357)
  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 4357)
  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: pMK349 (BSD-P2A-mAID-mCherry)
FEATURES             Location/Qualifiers
     source          1..4357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..90
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     82..99
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     83..99
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     109..128
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        109..127
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     152..168
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     153..169
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      184..193
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             190..585
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             598..654
                     /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             661..864
                     /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             871..1578
                     /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(1016..1034)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1598..1614)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1598..1614)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1651..1671)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1651..1669)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1690..1706)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1690..1706)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1703..1725)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1714..1730
                     /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(1738..1744,1745..1762,1763..1768))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1783..1804
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1921..1938)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2092..2680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2172..2191)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2851..3642,3643..3711))
                     /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     3474..3493
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3712..3816)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3843..4299
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3930..3949)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4140..4161
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
       61 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca
      121 ctatagggcg aattgggtac cgggcccccc ctcgaggtcg acggtatcga taagcttgat
      181 atcgccacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
      241 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
      301 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
      361 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
      421 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
      481 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
      541 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctcagg aagcggagct
      601 actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctagatct
      661 aaggagaaga gtgcttgtcc taaagatcca gccaaacctc cggccaaggc acaagttgtg
      721 ggatggccac cggtgagatc ataccggaag aacgtgatgg tttcctgcca aaaatcaagc
      781 ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg acggagcacc gtacttgagg
      841 aaaatcgatt tgaggatgta taaagctagc atggtgagca agggcgagga ggataacatg
      901 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
      961 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     1021 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     1081 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     1141 tccttccccg agggcttcaa ttgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     1201 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     1261 ggcaccaact tcccctccga cggccccgta atgcagtgtc gtaccatggg ctgggaggcc
     1321 tccactgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     1381 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     1441 cccgtgcagc tgcccggcgc ctacaacgtc gacatcaagt tggacatcct ttcccacaac
     1501 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     1561 atggacgagc tgtacaaggg cgctggtgca ggcgccggat ccactagttc tagagcggcc
     1621 gccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     1681 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1741 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     1801 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1861 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     1921 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1981 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2041 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2101 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2161 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2221 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2281 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2341 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2401 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2461 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2521 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2581 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2641 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2701 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     2761 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     2821 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2881 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2941 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3001 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3061 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3121 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3181 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3241 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3301 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3361 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3421 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3481 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3541 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3601 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3661 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3721 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3781 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3841 tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3901 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3961 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4021 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     4081 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4141 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4201 ataagggatt ttgccgattt cggcctattg gttaaaaaaa tgagctgatt taacaaaaat
     4261 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
     4321 caactgttgg gaagggcgat cggtgcgggc ctcttcg
//
