LOCUS       sequence-234559-        4774 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 4774)
  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 4774)
  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: pMK377 (Hygro-P2A-mCherry2)
FEATURES             Location/Qualifiers
     source          1..4774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..107
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     99..116
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     100..116
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     126..145
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        126..144
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     169..185
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     170..186
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             207..1229
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             207..1229
                     /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             1242..1298
                     /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             1305..2012
                     /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(1450..1468)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2032..2048)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2032..2048)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2085..2105)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2085..2103)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2124..2140)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2124..2140)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2137..2159)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2148..2164
                     /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(2172..2178,2179..2196,2197..2202))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2217..2238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2355..2372)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2526..3114)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2606..2625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3285..4076,4077..4145))
                     /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     3908..3927
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4146..4250)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4277..4733
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4364..4383)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4574..4595
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
       61 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc
      121 gcgcgtaata cgactcacta tagggcgaat tgggtaccgg gccccccctc gaggtcgacg
      181 gtatcgataa gcttgatatc gccaccatga aaaagcctga actcaccgcg acgtctgtcg
      241 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
      301 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
      361 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
      421 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
      481 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
      541 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
      601 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
      661 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
      721 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
      781 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
      841 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
      901 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
      961 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     1021 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     1081 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     1141 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     1201 gccccagcac tcgtccgagg gcaaaggaat caggaagcgg agctactaac ttcagcctgc
     1261 tgaagcaggc tggagacgtg gaggagaacc ctggacctag atctatggtg agcaagggcg
     1321 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
     1381 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1441 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1501 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     1561 actacttgaa gctgtccttc cccgagggct tcaattggga gcgcgtgatg aacttcgagg
     1621 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     1681 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag tgtcgtacca
     1741 tgggctggga ggcctccact gagcggatgt accccgagga cggcgccctg aagggcgaga
     1801 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1861 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcgacatc aagttggaca
     1921 tcctttccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     1981 actccaccgg cggcatggac gagctgtaca agggcgctgg tgcaggcgcc ggatccacta
     2041 gttctagagc ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta
     2101 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2161 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     2221 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     2281 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2341 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2401 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2461 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2521 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2581 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2641 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2701 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2761 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2821 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2881 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2941 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3001 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3061 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3121 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3181 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3241 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3301 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3361 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3421 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3481 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3541 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3601 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3661 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3721 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3781 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3841 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3901 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3961 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4021 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4081 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4141 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4201 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4261 cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4321 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4381 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4441 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4501 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4561 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     4621 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaaatgagct
     4681 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg
     4741 ccattcaggc tgcgcaactg ttgggaaggg cgat
//
