LOCUS       sequence-409725-        5734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains mAID-tag, mouse Ptbp1-specific homology arms and 
            blasticidin S resistance gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5734)
  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 5734)
  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: pML646
FEATURES             Location/Qualifiers
     source          1..5734
                     /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"
     regulatory      2279..2288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2285..2683
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    2705..2929
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2935..2968
                     /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(3546..3566)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3546..3564)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3585..3601)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3585..3601)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3598..3620)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3609..3625
                     /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(3633..3639,3640..3657,3658..3663))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3678..3699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3816..3833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3987..4575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4067..4086)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4746..5537,5538..5606))
                     /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     5369..5388
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5607..5711)
                     /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 gatctcgagc agctgaagct taccgctagc
     2281 caccatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac
     2341 aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga
     2401 cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact
     2461 cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat
     2521 cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga
     2581 tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg
     2641 gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacaat tcgagctcgg
     2701 taccctgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2761 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2821 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2881 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggt cgacgaagtt
     2941 cctattctct agaaagtata ggaacttcga acctgtgggc ctgcatcagc ttccaccatt
     3001 ccagaaaagc cactttaaaa agcagctgaa gtgaccttac agaccagaga ttttattttt
     3061 ttaaagagag atcagtttac ctgtttttaa aaaaattaaa tctagcccac tctgcagtgg
     3121 caggaaatgg cttggaccac agaggcccag tcaatcccag caccatgcct tgaggggctc
     3181 aggcgtgagg ccacagcggt gccccaaaca tagccgcttt ctgtgcctta caaaccagcg
     3241 tgcctcatgg ccgtacaccc accctctaag gccaccagtt gtctctagac ctgcaggtga
     3301 gccaggtggc ctatacatag gctccccaat gctgtgcaag agctggggcc actcccctac
     3361 acccttataa tcaagtgaca tgattctccc atgtccctgg ccagcccgga gggtccaggc
     3421 catcctcagc tgccgagcct cattgtgacc tttgtattct attccgtgtg gtcacagaca
     3481 cctgtgccta gcaatatttc cacttgacca aatacccgaa tcctaggtgt acccagcttt
     3541 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     3601 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3661 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3721 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3781 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3841 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3901 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3961 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4021 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4081 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4141 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4201 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4261 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4321 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4381 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4441 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4501 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4561 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4621 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4681 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4741 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4801 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4861 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4921 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4981 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5041 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5101 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5161 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5221 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5281 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5341 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5401 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5461 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5521 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5581 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5641 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5701 ggggttccgc gcacatttcc ccgaaaagtg ccac
//
