LOCUS       sequence-409729-        5904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains mAID-tag, mouse Ptbp1-specific homology arms and puromycin 
            resistance gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5904)
  AUTHORS   Kainov Y, Zhuravskaya A, Makeyev EV
  TITLE     Protocol for auxin-inducible depletion of the RNA-binding protein 
            PTBP1 in mouse embryonic stem cells.
  JOURNAL   STAR Protoc. 2023 Oct 18;4(4):102644. doi: 
            10.1016/j.xpro.2023.102644.
  PUBMED    37862173
REFERENCE   2  (bases 1 to 5904)
  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: "STAR 
            Protoc."; date: "2023-10-18"; volume: "4(4):102644. doi"; pages: " 
            10.1016/j.xpro.2023.102644"
COMMENT     Sequence Label: pML60
FEATURES             Location/Qualifiers
     source          1..5904
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward 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"
     CDS             1254..1457
                     /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"
     protein_bind    1467..1500
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    1603..1724
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1640..1659)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1694..1713
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1736..2235
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1771..1794)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2168..2187
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2269..2868
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2269..2288)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2765..2785
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2875..3099
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3105..3138
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(3716..3736)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3716..3734)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3755..3771)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3755..3771)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3768..3790)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3779..3795
                     /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(3803..3809,3810..3827,3828..3833))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3848..3869
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3986..4003)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4157..4745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4237..4256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4916..5707,5708..5776))
                     /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     5539..5558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5777..5881)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcgc
      661 attacactgt ccaagcatca gagtgtgcag ctgcctcggg agggtcagga ggaccagggc
      721 ctgaccaagg actatggcag ctccccgctg caccgcttca agaaaccagg ctccaagaac
      781 ttccagaaca tctttccacc ctcagctacc ctgcacctct ccaacatccc gtaagtcagc
      841 cagaggtctt gtttgtgggt gggccacgaa gggctcctga tctcccccac cccatccccg
      901 tgtgagccca ggccgatggc tcccgtctgt ctctgcaggc cctctgtgtc agaggacgac
      961 ctcaagagcc tcttctccag caacggtggt gtggtcaaag gcttcaagtt cttccagtga
     1021 gtaaagccct gcctttcgtc ctccaccctt aaccctaccc caaacctgca ggtttagctg
     1081 actacccccc actccacccc tgcaggaagg accgcaagat ggcactgatc cagatgggct
     1141 ctgtggagga ggctgtgcag gcgctgattg aactgcacaa ccatgacctg ggcgagaacc
     1201 accacctgcg agtgtccttt tccaagtcca ccatcggatc cggtgcaggc gccaaggaga
     1261 agagtgcttg tcctaaagat ccagccaaac ctccggccaa ggcacaagtt gtgggatggc
     1321 caccggtgag atcataccgg aagaacgtga tggtttcctg ccaaaaatca agcggtggcc
     1381 cggaggcggc ggcgttcgtg aaggtatcaa tggacggagc accgtacttg aggaaaatcg
     1441 atttgaggat gtataaataa tctagcgaag ttcctattct ctagaaagta taggaacttc
     1501 gatatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     1561 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     1621 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     1681 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaagaatt ctaccgggta
     1741 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact
     1801 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa
     1861 ccgactccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag
     1921 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc
     1981 actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc
     2041 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg
     2101 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga
     2161 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca
     2221 tctccgggcc tttcgacctg catccatcta gatccaccgg agcttaccat gaccgagtac
     2281 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     2341 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     2401 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     2461 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     2521 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     2581 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     2641 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     2701 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     2761 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     2821 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgagg taccctgtgc
     2881 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     2941 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3001 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3061 acaatagcag gcatgctggg gatgcggtgg gctctatggt cgacgaagtt cctattctct
     3121 agaaagtata ggaacttcga acctgtgggc ctgcatcagc ttccaccatt ccagaaaagc
     3181 cactttaaaa agcagctgaa gtgaccttac agaccagaga ttttattttt ttaaagagag
     3241 atcagtttac ctgtttttaa aaaaattaaa tctagcccac tctgcagtgg caggaaatgg
     3301 cttggaccac agaggcccag tcaatcccag caccatgcct tgaggggctc aggcgtgagg
     3361 ccacagcggt gccccaaaca tagccgcttt ctgtgcctta caaaccagcg tgcctcatgg
     3421 ccgtacaccc accctctaag gccaccagtt gtctctagac ctgcaggtga gccaggtggc
     3481 ctatacatag gctccccaat gctgtgcaag agctggggcc actcccctac acccttataa
     3541 tcaagtgaca tgattctccc atgtccctgg ccagcccgga gggtccaggc catcctcagc
     3601 tgccgagcct cattgtgacc tttgtattct attccgtgtg gtcacagaca cctgtgccta
     3661 gcaatatttc cacttgacca aatacccgaa tcctaggtgt acccagcttt tgttcccttt
     3721 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3781 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3841 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     3901 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3961 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4021 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4081 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4141 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4201 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4261 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4321 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4381 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4441 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4501 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4561 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4621 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4681 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4741 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4801 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4861 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4921 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4981 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5041 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5101 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5161 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5221 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5281 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5341 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5401 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5461 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5521 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5581 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5641 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5701 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5761 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5821 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5881 gcacatttcc ccgaaaagtg ccac
//
