LOCUS       sequence-235391-        4783 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 4783)
  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 4783)
  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: pMK376 (Hygro-P2A-mClover)
FEATURES             Location/Qualifiers
     source          1..4783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..111
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     103..120
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     104..120
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     130..149
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        130..148
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     173..189
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     174..190
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             211..1233
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             211..1233
                     /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             1246..1302
                     /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             join(1309..1311,1312..1314,1315..2025)
                     /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(1354..1375)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1962..1983
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2045..2061)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2045..2061)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2098..2118)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2098..2116)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2137..2153)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2137..2153)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2150..2172)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2161..2177
                     /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(2185..2191,2192..2209,2210..2215))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2230..2251
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2368..2385)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2539..3127)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2619..2638)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3298..4089,4090..4158))
                     /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     3921..3940
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4159..4263)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4290..4746
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4377..4396)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4587..4608
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
       61 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
      121 gagcgcgcgt aatacgactc actatagggc gaattgggta ccgggccccc cctcgaggtc
      181 gacggtatcg ataagcttga tatcgccacc atgaaaaagc ctgaactcac cgcgacgtct
      241 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
      301 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
      361 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
      421 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
      481 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
      541 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
      601 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
      661 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
      721 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
      781 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
      841 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
      901 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
      961 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     1021 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     1081 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     1141 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     1201 cgacgcccca gcactcgtcc gagggcaaag gaatcaggaa gcggagctac taacttcagc
     1261 ctgctgaagc aggctggaga cgtggaggag aaccctggac ctagatctat ggtgagcaag
     1321 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1381 ggccacaagt tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg caagctgacc
     1441 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1501 ttcggctacg gcgtggcctg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1561 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcttt caaggacgac
     1621 ggtacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1681 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1741 aacttcaaca gccacaacgt ctatatcacg gccgacaagc agaagaacgg catcaaggct
     1801 aacttcaaga tccgccacaa cgttgaggac ggcagcgtgc agctcgccga ccactaccag
     1861 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagccat
     1921 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1981 gtgaccgccg ccgggattac acatggcatg gacgagctgt acaagggcgc tggtgcaggc
     2041 gccggatcca ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct
     2101 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     2161 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     2221 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     2281 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     2341 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2401 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2461 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2521 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2581 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2641 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2701 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2761 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2821 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2881 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2941 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3001 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3061 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3121 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3181 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3241 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3301 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3361 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3421 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     3481 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3541 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3601 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3661 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3721 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3781 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3841 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3901 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3961 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4021 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4081 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4141 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4201 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4261 gcgcacattt ccccgaaaag tgccacctga cgcgccctgt agcggcgcat taagcgcggc
     4321 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4381 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4441 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4501 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4561 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4621 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4681 aaaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     4741 caatttccat tcgccattca ggctgcgcaa ctgttgggaa ggg
//
