LOCUS       sequence-234556-        3643 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 3643)
  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 3643)
  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: pMK347 (BSD-P2A-mAID)
FEATURES             Location/Qualifiers
     source          1..3643
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(77..96)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     287..308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     572..594
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     586..603
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     587..603
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     613..632
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        613..631
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     656..672
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     657..673
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      688..697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             694..1089
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1102..1158
                     /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             1165..1368
                     /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"
     primer_bind     complement(1388..1404)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1388..1404)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1441..1461)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1441..1459)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1480..1496)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1480..1496)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1493..1515)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1504..1520
                     /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(1528..1534,1535..1552,1553..1558))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1573..1594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1711..1728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1882..2470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1962..1981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2641..3432,3433..3501))
                     /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     3264..3283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3502..3606)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(3633..3643,1..446)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
       61 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
      121 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
      181 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
      241 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
      301 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
      361 ccgatttcgg cctattggtt aaaaaaatga gctgatttaa caaaaattta acgcgaattt
      421 taacaaaata ttaacgctta caatttccat tcgccattca ggctgcgcaa ctgttgggaa
      481 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
      541 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
      601 agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag
      661 gtcgacggta tcgataagct tgatatcgcc accatggcca agcctttgtc tcaagaagaa
      721 tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac
      781 agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat
      841 cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca
      901 gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc
      961 tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgaag
     1021 gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg
     1081 tgggagggct caggaagcgg agctactaac ttcagcctgc tgaagcaggc tggagacgtg
     1141 gaggagaacc ctggacctag atctaaggag aagagtgctt gtcctaaaga tccagccaaa
     1201 cctccggcca aggcacaagt tgtgggatgg ccaccggtga gatcataccg gaagaacgtg
     1261 atggtttcct gccaaaaatc aagcggtggc ccggaggcgg cggcgttcgt gaaggtatca
     1321 atggacggag caccgtactt gaggaaaatc gatttgagga tgtataaagg cgctggtgca
     1381 ggcgccggat ccactagttc tagagcggcc gccaccgcgg tggagctcca gcttttgttc
     1441 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     1501 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     1561 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     1621 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     1681 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     1741 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     1801 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1861 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1921 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1981 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2041 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2101 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2161 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2221 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2281 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2341 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2401 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2461 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     2521 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     2581 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2641 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2701 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2761 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     2821 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2881 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2941 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3001 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3061 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3121 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3181 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3241 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3301 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3361 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3421 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3481 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     3541 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3601 tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgt
//
