LOCUS       sequence-426333-        8753 bp    DNA     circular SYN 14-APR-2025
DEFINITION  EF1-OsTIR1(F74G) (PiggyBac).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8753)
  AUTHORS   Hatoyama Y, Islam M, Bond AG, Hayashi KI, Ciulli A, Kanemaki MT
  TITLE     Combination of AID2 and BromoTag expands the utility of degron-based
            protein knockdowns.
  JOURNAL   EMBO Rep. 2024 Sep;25(9):4062-4077. doi: 10.1038/s44319-024-00224-4.
            Epub 2024 Aug 23.
  PUBMED    39179892
REFERENCE   2  (bases 1 to 8753)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s44319-024-00224-4"; journalName: "EMBO Rep"; date: 
            "2024-09"; volume: "25"; issue: "9"; pages: "4062-4077"
COMMENT     Sequence Label: pMK444 (EF1-OsTIR1(F74G) Puro)
FEATURES             Location/Qualifiers
     source          1..8753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1153..2335
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1384..2326
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2283..2303
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     polyA_signal    4276..4397
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4313..4332)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4367..4386
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    4424..4457
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4518..5017
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4553..4576)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4950..4969
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5057..5656
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5057..5076)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    6213..6246
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   6452..6514
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6565..6585)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6565..6583)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6604..6620)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6604..6620)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6617..6639)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6628..6644
                     /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(6652..6658,6659..6676,6677..6682))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6697..6718
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6835..6852)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7006..7594)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7086..7105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7765..8556,8557..8625))
                     /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     8388..8407
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8626..8730)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagtaat gagtaattca tacaaaagga
     1021 ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa ctcccactaa cgtagaaccc
     1081 agagatcgct gcgttcccgc cccctcaccc gcccgctctc gtcatcactg aggtggagaa
     1141 gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     1201 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
     1261 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     1321 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     1381 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc
     1441 gtgccttgaa ttacttccac gcccctggct gcagtacgtg attcttgatc ccgagcttcg
     1501 ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc
     1561 ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg
     1621 cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc
     1681 gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat
     1741 ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc
     1801 gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg
     1861 gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct
     1921 ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg
     1981 gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag
     2041 gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc
     2101 gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga
     2161 ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc
     2221 ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat
     2281 tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgacgcta
     2341 gcccggactc agatctcgag ctcaagcttc gaattcgagc tcggtacctc gcgaatgcat
     2401 ctagatagcg ctgccaccat gacatacttt cctgaagagg tcgtcgaaca catttttagc
     2461 ttcctgcctg cacagagaga tagaaacaca gtgagcctgg tctgcaaagt gtggtacgag
     2521 atcgaacgcc tgagccggag aggagtgttc gtcggcaact gctatgctgt gagagcaggc
     2581 agggtcgccg ctaggtttcc aaatgtgcgc gcactgaccg tcaaggggaa accccacggc
     2641 gccgacttta acctggtgcc ccctgattgg ggaggatacg ccggcccttg gatcgaggca
     2701 gccgctcgcg gctgtcatgg actggaggaa ctgcgcatga agcgaatggt ggtctctgac
     2761 gaaagtctgg agctgctggc tcggagcttc cctaggtttc gcgcactggt gctgatttct
     2821 tgcgaaggct tcagcaccga tggactggca gccgtggcct cccactgtaa gctgctgcgg
     2881 gagctggacc tccaggagaa tgaagtggag gatagaggcc ccagatggct gtcttgcttc
     2941 ccagactcat gtaccagcct ggtgtccctg aactttgcct gcatcaaagg cgaagtgaat
     3001 gctgggtccc tggagcggct ggtctcaaga agccccaacc tgaggtctct gcggctgaac
     3061 cggagcgtga gcgtggacac tctggctaag attctgctga gaacccctaa cctggaggat
     3121 ctgggaaccg gcaatctgac agacgatttc cagacagaat cctactttaa actgacttct
     3181 gccctggaga agtgtaaaat gctgaggagt ctgtcaggat tctgggatgc ttcacccgtg
     3241 tgcctgagct ttatctaccc tctgtgtgca cagctgacag gcctgaacct gagctatgca
     3301 ccaaccctgg acgccagtga tctgacaaag atgatctcac gctgcgtgaa actccagcga
     3361 ctgtgggtgc tggactgtat ttccgataag gggctccagg tggtcgccag ctcctgcaag
     3421 gacctccagg agctgagagt gttcccatct gatttttacg tggccggata tagtgctgtc
     3481 actgaggaag gcctggtggc agtctcactg ggatgcccaa agctgaacag cctgctgtat
     3541 ttctgtcatc agatgactaa tgctgcactg gtgaccgtcg ccaagaactg ccctaatttc
     3601 acccgatttc ggctgtgtat tctggaacca ggcaaacccg acgtggtcac atcccagcca
     3661 ctggatgaag ggtttggagc tatcgtgaga gagtgcaagg gactccagag gctgagcatt
     3721 tccggcctgc tgacagacaa agtgttcatg tacatcggca agtatgctaa gcagctggag
     3781 atgctgagca ttgcatttgc cggagactcc gataagggca tgatgcacgt gatgaacggg
     3841 tgtaagaatc tgcgaaaact ggaaatccgg gacagccctt tcggggatgc cgctctgctg
     3901 ggaaactttg ccagatacga gacaatgagg agcctgtgga tgtctagttg caatgtgact
     3961 ctgaagggct gtcaggtcct ggctagtaaa atgcctatgc tgaacgtgga agtcattaat
     4021 gagcgggacg ggtctaacga aatggaggaa aatcatggcg acctgccaaa ggtggagaaa
     4081 ctgtatgtgt atcggaccac cgcaggggca agagatgatg ctcccaactt tgtgaagatt
     4141 ctgtgaggat ccaccggatc tagataactg atcataatca gccataccac atttgtagag
     4201 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     4261 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4321 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4381 ctcatcaatg tatcttaagc gatcatattc aataaccctt aatataactt cgtataatgt
     4441 atgctatacg aagttattag gtctgaagag gagtttacgt ccagccaagc ttaggatctc
     4501 gacctcgaaa ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc
     4561 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc
     4621 cacatccacc ggtaggcgcc aaccgactcc gttctttggt ggccccttcg cgccaccttc
     4681 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
     4741 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
     4801 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
     4861 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc
     4921 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc
     4981 cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcatccatc tagatctcga
     5041 gcagctgaag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     5101 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     5161 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     5221 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     5281 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     5341 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     5401 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca
     5461 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     5521 ggtgcccgcc ttcctggaga cctccgcgcc ccacaacctc cccttctacg agcggctcgg
     5581 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     5641 caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg
     5701 accccatgca tcgatgatat cagatccccg ggatgcagaa attgatgatc tattaaacaa
     5761 taaagatgtc cactaaaatg gaagtttttc ctgtcatact ttgttaagaa gggtgagaac
     5821 agagtaccta cattttgaat ggaaggattg gagctacggg ggtgggggtg gggtgggatt
     5881 agataaatgc ctgctcttta ctgaaggctc tttactattg ctttatgata atgtttcata
     5941 gttggatatc ataatttaaa caagcaaaac caaattaagg gccagctcat tcctcccact
     6001 catgatctat agatctatag atctctcgtg ggatcattgt ttttctcttg attcccactt
     6061 tgtggttcta agtactgtgg tttccaaatg tgtcagtttc atagcctgaa gaacgagatc
     6121 agcagcctct gttccacata cacttcattc tcagtattgt tttgccaagt tctaattcca
     6181 tcagaagctg gtcgagatcc ggaaccctta atataacttc gtataatgta tgctatacga
     6241 agttattagg tccctcgaag aggttcacta gctagttaaa agttttgtta ctttatagaa
     6301 gaaattttga gtttttgttt ttttttaata aataaataaa cataaataaa ttgtttgttg
     6361 aatttattat tagtatgtaa gtgtaaatat aataaaactt aatatctatt caaattaata
     6421 aataaacctc gatatacaga ccgataaaac acatgcgtca attttacgca tgattatctt
     6481 taacgtacgt cacaatatga ttatctttct agggttaatc tagtatacgc gtatgcggcc
     6541 gcttaattaa tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     6601 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     6661 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     6721 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6781 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6841 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6901 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6961 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     7021 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     7081 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     7141 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     7201 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     7261 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     7321 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     7381 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     7441 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     7501 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     7561 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7621 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7681 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7741 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7801 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7861 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7921 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7981 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     8041 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     8101 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     8161 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     8221 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     8281 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     8341 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     8401 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     8461 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     8521 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8581 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     8641 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     8701 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
