LOCUS       sequence-457074-        7101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology repair plasmid for endogenous tagging of AP1muA at the 
            C-terminus with TurboID and a V5 epitope tag. Contains a G418 
            resistance cassette for selection of edited cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7101)
  AUTHORS   Stockhammer A, Spalt C, Klemt A, Benz LS, Harel S, Natalia V, Wiench
            L, Freund C, Kuropka B, Bottanelli F
  TITLE     When less is more - a fast TurboID knock-in approach for 
            high-sensitivity endogenous interactome mapping.
  JOURNAL   J Cell Sci. 2024 Aug 15;137(16):jcs261952. doi: 10.1242/jcs.261952. 
            Epub 2024 Aug 28.
  PUBMED    39056144
REFERENCE   2  (bases 1 to 7101)
  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: "J Cell 
            Sci."; date: "2024-08-15"; volume: "137(16):jcs261952. doi"; pages: 
            " 10.1242/jcs.261952. Epub 2024 Aug 28"
COMMENT     Sequence Label: AP1muA-TurboID-polyA-G418 HR
FEATURES             Location/Qualifiers
     source          1..7101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(706..730)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(830..1621,1622..1690))
                     /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"
     promoter        complement(1691..1795)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1894..1916
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1908..1925
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1909..1925
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3119..4078
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             4079..4120
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    4247..4368
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4284..4303)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4338..4357
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4394..4751
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4394..4585
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4417..4437)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4602..4737
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4786..5580
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4786..5580
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4840..4859)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5450..5469
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5768..5787)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(6914..6930)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6914..6930)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6927..6949)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctctatt caacaaagcc gccgtcccgt caagtcagcg
      661 taaatgggta gggggcttca aatcgtcctc gtgataccaa ttcggagcct gcttttttgt
      721 acaaacttgt tgataatggc aattcaagga tcttcaccta gatcctttta aattaaaaat
      781 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
      841 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
      901 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
      961 tgataccgcg agagccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     1021 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     1081 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     1141 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     1201 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     1261 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     1321 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     1381 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     1441 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     1501 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     1561 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     1621 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     1681 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     1741 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     1801 ttccccgaaa agtgccagat acctgaaaca aaacccatcg tacggccaag gaagtctcca
     1861 ataactgtga tccaccacaa gcgccagggt tttcccagtc acgacgttgt aaaacgacgg
     1921 ccagtcatgc ataatccgca cgcatctgga ataaggaagt gccattccgc ctgaccttgg
     1981 ggcagcacag ggccgagctt tggctcccca gcacaggggc tcatgctccc ctccctggac
     2041 tggggtgagt ggtcagtctc tgcccatcgc cactagggcc agagctgttg gctaagctgg
     2101 ggcagggcct aggcctgagc gcctcccccg tctgctcccc agggcggcaa ggagtacctg
     2161 atgcgggccc acttcggcct gcctagtgtg gaggccgaag acaaggaggg caagcccccg
     2221 atcagtgtca agttcgagat cccttacttc actacctccg gcatccaggt acgtaaggcc
     2281 caggcggccg gggcccagaa cagggacaga ggccgcaggt gacagatatc aatgcagcca
     2341 gcttgggaaa gggtgtcagt cagctgcaat caggaccctg ctgtgctgtg ggcctctgcc
     2401 tccccacaga cagggtgagg ccaggctgcc tctgctcagc cctgacccct gagctcaaag
     2461 ctcactcatt ctgccccaaa ctagcatttg aggctctgcc gtccactcat gccccatggg
     2521 acctcttttc cctggcaccc cctcaaaagc ccacagaggg aggagaggct cccagagagc
     2581 caggggcctt ctggccgggc cccccaagca ggctgctgag gccgtgtctg caagccacat
     2641 cctggctgct caccacttcc agaggcggct ctgccctccc acacatttgc atggtccaga
     2701 caaatccctt tcacccccct gaggcctgtg ctcatcctgt cgtcatggcc tcccctgcca
     2761 gccccaggct gccccagcag gaaagattaa ggggggacca acagggcggg agcctgcggt
     2821 gctcagggcc ctgctacctg tgccccaggt gcgctacctg aagatcattg agaagagtgg
     2881 gtaccaggcc ctgccctggg tgcgttatat cacgcagaat ggaggtgagt gaggccctac
     2941 ccgtggggtg gggttgtact gaggggtcct ctgtggctgc ccccagggtg gagccatcgg
     3001 gtcgggtccc gaaagcaggg agggtgcaga gcccagcatg acgcctccct cctgtctcct
     3061 agattaccag ctccggaccc agggatcagg gtcaggctct ggttccggct caggatccat
     3121 gaaagacaat actgtgcctc tgaagctgat cgctctcctg gctaatggcg agttccatag
     3181 tggcgaacag ctgggagaaa ccctgggcat gtccagggcc gctatcaaca agcacattca
     3241 gactctgcgc gactggggcg tggacgtgtt caccgtgccc ggaaagggct actctctgcc
     3301 cgagcctatc ccgctgctga acgctaaaca gattctggga cagctggacg gcgggagcgt
     3361 ggcagtcctg cctgtggtcg actccaccaa tcagtacctg ctggatcgaa tcggcgagct
     3421 gaagagtggg gatgcttgca ttgcagaata tcagcaggca gggagaggaa gcagagggag
     3481 gaaatggttc tctccttttg gagctaacct gtacctgagt atgttttggc gcctgaagcg
     3541 gggaccagca gcaatcggcc tgggcccggt catcggaatt gtcatggcag aagcgctgcg
     3601 aaagctggga gcagacaagg tgcgagtcaa atggcccaat gacctgtatc tgcaggatag
     3661 aaagctggca ggcatcctgg tggagctggc cggaataaca ggcgatgctg cacagatcgt
     3721 cattggcgcc gggattaacg tggctatgag gcgcgtggag gaaagcgtgg tcaatcaggg
     3781 ctggatcaca ctgcaggaag cagggattaa cctggacagg aatactctgg ccgctacgct
     3841 gatccgagag ctgcgggcag ccctggaact gttcgagcag gaaggcctgg ctccatatct
     3901 gccacggtgg gagaagctgg ataacttcat caatagaccc gtgaagctga tcattgggga
     3961 caaagagatt ttcgggatta gccgggggat tgataaacag ggagccctgc tgctggaaca
     4021 ggacggagtt atcaaaccct ggatgggcgg agaaatcagt ctgcggtctg ccgaaaaggg
     4081 caagcccatc cccaaccccc tgctgggcct ggacagcacc tgagctagcc cgcgactcta
     4141 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     4201 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     4261 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4321 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg
     4381 taaggcggcc gccctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     4441 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4501 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4561 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4621 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4681 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4741 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4801 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4861 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4921 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     4981 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     5041 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     5101 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     5161 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     5221 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     5281 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     5341 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     5401 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     5461 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     5521 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5581 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5641 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5701 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     5761 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     5821 aaagacagaa taaagaattc ggggctgtcg cagccaacac cccagcctcg gagctcctgg
     5881 tggcagcacc aggggacaca cctgccaaac ccaccagatg gaggggccct ccctggtctc
     5941 tggccaccct cccagcctct gcccagggac ccctgccttc cccaggccat ctgctctgcc
     6001 gtcgacactc gtctcagaag cccctttccc agaagaggct ggtcttcaag aagtctcgtt
     6061 tctttgcccc tgaagtcagt ttcaggggaa ggatgtgaaa tttttccgtg tagaggttac
     6121 agccttttat gctgttgagc tcccaggtac caaaaagctt ggccaacgct tgccagccag
     6181 ccagctgcag gtggcatctg ccacgaagga agcgccagcc tcgccaggcc agcaggggcg
     6241 tcgttttgtt gccattttgt tgaacgttat gggtttatgg gtgttcctgg aacttgtctt
     6301 tgtgcattcg ttgctgtttg tgttaccctc actgtcccca tgtcccaccc acgtcctacg
     6361 gcactcagga agcacttggt gaggacgagc cctcaccctt cttgtcttcc ttcccggcag
     6421 cgcccgcagc gggccattta cacgtcgagg ctggcacctg gcgcgctcgg gggccactgt
     6481 agcgtctgcc tgctccctgg actcgcaggc ctcgcctgtg gcgccttccc agggccagcc
     6541 tgggtcacga gatgctgtca ctcagccaga tcagtattga cccaccaggg gaggtggggt
     6601 ttggtgagag acgccagcct cagacttttt cccactgagg gtccagagag cggggccacg
     6661 tgtcacccac gtctgcgctt ggtcacccgt cctccccacc ctgtgtgtgt ttatgtcata
     6721 gttacattaa attccattca ttgaatactc cacagccttc ctctgatgca gcgtggtccc
     6781 aactcctcct gcgtgaccac agggcttgcc tggttttgtc gccgaccttc cctgtggccc
     6841 cacgaggtgt caccagcagg ctaggtggag gctcagtgat gataagtctg cgatggtgga
     6901 tgcatgtgtc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc agagggcaca
     6961 atcctattcc gcgctatccg acaatctcca agacattagg tggagttcag ttcggcgtat
     7021 ggcatatgtc gctggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     7081 aaaggccgcg ttgctggcgt t
//
