LOCUS       sequence-409728-        6350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains mAID-tag, mouse Ptbp1-specific homology arms and hygromycin
            b resistance gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6350)
  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 6350)
  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: pML58
FEATURES             Location/Qualifiers
     source          1..6350
                     /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             2296..3315
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2296..3315
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     polyA_signal    3321..3545
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3551..3584
                     /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(4162..4182)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4162..4180)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4201..4217)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4201..4217)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4214..4236)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4225..4241
                     /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(4249..4255,4256..4273,4274..4279))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4294..4315
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4432..4449)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4603..5191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4683..4702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5362..6153,6154..6222))
                     /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     5985..6004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6223..6327)
                     /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 atggatagat ccggaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     2341 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     2401 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     2461 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     2521 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     2581 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     2641 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     2701 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     2761 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     2821 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     2881 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     2941 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     3001 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     3061 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     3121 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     3181 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     3241 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     3301 agggcaaagg aataggtacc ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3361 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3421 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3481 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3541 tatggtcgac gaagttccta ttctctagaa agtataggaa cttcgaacct gtgggcctgc
     3601 atcagcttcc accattccag aaaagccact ttaaaaagca gctgaagtga ccttacagac
     3661 cagagatttt atttttttaa agagagatca gtttacctgt ttttaaaaaa attaaatcta
     3721 gcccactctg cagtggcagg aaatggcttg gaccacagag gcccagtcaa tcccagcacc
     3781 atgccttgag gggctcaggc gtgaggccac agcggtgccc caaacatagc cgctttctgt
     3841 gccttacaaa ccagcgtgcc tcatggccgt acacccaccc tctaaggcca ccagttgtct
     3901 ctagacctgc aggtgagcca ggtggcctat acataggctc cccaatgctg tgcaagagct
     3961 ggggccactc ccctacaccc ttataatcaa gtgacatgat tctcccatgt ccctggccag
     4021 cccggagggt ccaggccatc ctcagctgcc gagcctcatt gtgacctttg tattctattc
     4081 cgtgtggtca cagacacctg tgcctagcaa tatttccact tgaccaaata cccgaatcct
     4141 aggtgtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
     4201 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4261 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4321 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4381 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4441 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4501 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4561 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4621 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4681 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4741 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4801 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4861 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4921 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4981 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5041 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5101 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5161 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5221 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5281 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5341 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5401 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5461 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5521 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5581 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5641 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5701 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5761 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5821 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5881 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5941 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6001 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6061 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6121 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6181 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6241 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6301 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
//
