LOCUS       sequence-235302-        4270 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 4270)
  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 4270)
  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: pMK344 (Hygro-P2A-mAID)
FEATURES             Location/Qualifiers
     source          1..4270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..71
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     63..80
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     64..80
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     90..109
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        90..108
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     133..149
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     134..150
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             171..1193
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             171..1193
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1206..1262
                     /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             1269..1472
                     /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(1492..1508)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1492..1508)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1545..1565)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1545..1563)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1584..1600)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1584..1600)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1597..1619)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1608..1624
                     /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(1632..1638,1639..1656,1657..1662))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1677..1698
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1815..1832)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1986..2574)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2066..2085)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2745..3536,3537..3605))
                     /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     3368..3387
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3606..3710)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3737..4193
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3824..3843)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4034..4055
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
       61 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      121 ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgccacc atgaaaaagc
      181 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
      241 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
      301 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
      361 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
      421 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
      481 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
      541 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
      601 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
      661 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
      721 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
      781 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
      841 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
      901 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
      961 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     1021 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     1081 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     1141 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatcaggaa
     1201 gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     1261 ctagatctaa ggagaagagt gcttgtccta aagatccagc caaacctccg gccaaggcac
     1321 aagttgtggg atggccaccg gtgagatcat accggaagaa cgtgatggtt tcctgccaaa
     1381 aatcaagcgg tggcccggag gcggcggcgt tcgtgaaggt atcaatggac ggagcaccgt
     1441 acttgaggaa aatcgatttg aggatgtata aaggcgctgg tgcaggcgcc ggatccacta
     1501 gttctagagc ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta
     1561 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     1621 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     1681 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1741 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1801 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1861 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1921 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1981 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2041 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2101 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2161 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2221 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2281 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2341 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2401 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2461 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2521 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2581 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2641 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2701 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2761 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2821 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2881 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2941 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3001 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3061 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3121 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3181 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3241 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3301 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3361 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3421 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3481 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3541 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3601 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3661 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3721 cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3781 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3841 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3901 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3961 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4021 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     4081 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaaatgagct
     4141 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg
     4201 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     4261 cagctggcga
//
