LOCUS       sequence-456978-        7014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology repair plasmid for endogenous tagging of AP1muA at the 
            C-terminus with HaloTag and an ALFA 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 7014)
  AUTHORS   Stockhammer A, Adarska P, Natalia V, Heuhsen A, Klemt A, Bregu G, 
            Harel S, Rodilla-Ramirez C, Spalt C, Ozsoy E, Leupold P, Grindel A, 
            Fox E, Mejedo JO, Zehtabian A, Ewers H, Puchkov D, Haucke V, 
            Bottanelli F
  TITLE     ARF1 compartments direct cargo flow via maturation into recycling 
            endosomes.
  JOURNAL   Nat Cell Biol. 2024 Oct 4. doi: 10.1038/s41556-024-01518-4.
  PUBMED    39367144
REFERENCE   2  (bases 1 to 7014)
  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: "Nat Cell 
            Biol. 2024 Oct 4. doi: 10.1038/s41556-024-01518-4."
COMMENT     Sequence Label: AP1muA-Halo-ALFA-polyA-G418 HR
FEATURES             Location/Qualifiers
     source          1..7014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    48..169
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(85..104)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     139..158
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     enhancer        195..386
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(218..238)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             566..1360
                     /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             566..1360
                     /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(620..639)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1230..1249
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1548..1567)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(2694..2710)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2694..2710)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2707..2729)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2882..3470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2962..2981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3587..3611)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3711..4502,4503..4571))
                     /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     4334..4353
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4572..4676)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4775..4797
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4789..4806
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4790..4806
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             6000..6890
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             6894..6932
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
ORIGIN
        1 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
       61 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
      121 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
      181 gtaaggcggc cgccctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
      241 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
      301 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
      361 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
      421 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      481 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
      541 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
      601 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
      661 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
      721 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
      781 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
      841 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
      901 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
      961 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     1021 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     1081 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     1141 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     1201 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     1261 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     1321 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     1381 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     1441 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     1501 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     1561 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaagaattc
     1621 ggggctgtcg cagccaacac cccagcctcg gagctcctgg tggcagcacc aggggacaca
     1681 cctgccaaac ccaccagatg gaggggccct ccctggtctc tggccaccct cccagcctct
     1741 gcccagggac ccctgccttc cccaggccat ctgctctgcc gtcgacactc gtctcagaag
     1801 cccctttccc agaagaggct ggtcttcaag aagtctcgtt tctttgcccc tgaagtcagt
     1861 ttcaggggaa ggatgtgaaa tttttccgtg tagaggttac agccttttat gctgttgagc
     1921 tcccaggtac caaaaagctt ggccaacgct tgccagccag ccagctgcag gtggcatctg
     1981 ccacgaagga agcgccagcc tcgccaggcc agcaggggcg tcgttttgtt gccattttgt
     2041 tgaacgttat gggtttatgg gtgttcctgg aacttgtctt tgtgcattcg ttgctgtttg
     2101 tgttaccctc actgtcccca tgtcccaccc acgtcctacg gcactcagga agcacttggt
     2161 gaggacgagc cctcaccctt cttgtcttcc ttcccggcag cgcccgcagc gggccattta
     2221 cacgtcgagg ctggcacctg gcgcgctcgg gggccactgt agcgtctgcc tgctccctgg
     2281 actcgcaggc ctcgcctgtg gcgccttccc agggccagcc tgggtcacga gatgctgtca
     2341 ctcagccaga tcagtattga cccaccaggg gaggtggggt ttggtgagag acgccagcct
     2401 cagacttttt cccactgagg gtccagagag cggggccacg tgtcacccac gtctgcgctt
     2461 ggtcacccgt cctccccacc ctgtgtgtgt ttatgtcata gttacattaa attccattca
     2521 ttgaatactc cacagccttc ctctgatgca gcgtggtccc aactcctcct gcgtgaccac
     2581 agggcttgcc tggttttgtc gccgaccttc cctgtggccc cacgaggtgt caccagcagg
     2641 ctaggtggag gctcagtgat gataagtctg cgatggtgga tgcatgtgtc atggtcatag
     2701 ctgtttcctg tgtgaaattg ttatccgctc agagggcaca atcctattcc gcgctatccg
     2761 acaatctcca agacattagg tggagttcag ttcggcgtat ggcatatgtc gctggaaaga
     2821 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2881 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2941 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3001 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3061 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3121 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3181 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3241 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3301 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3361 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3421 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3481 tgatcttttc tacggggtct gacgctctat tcaacaaagc cgccgtcccg tcaagtcagc
     3541 gtaaatgggt agggggcttc aaatcgtcct cgtgatacca attcggagcc tgcttttttg
     3601 tacaaacttg ttgataatgg caattcaagg atcttcacct agatcctttt aaattaaaaa
     3661 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3721 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3781 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3841 atgataccgc gagagccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3901 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3961 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4021 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4081 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4141 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4201 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4261 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4321 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4381 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4441 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4501 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4561 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4621 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4681 tttccccgaa aagtgccaga tacctgaaac aaaacccatc gtacggccaa ggaagtctcc
     4741 aataactgtg atccaccaca agcgccaggg ttttcccagt cacgacgttg taaaacgacg
     4801 gccagtcatg cataatccgc acgcatctgg aataaggaag tgccattccg cctgaccttg
     4861 gggcagcaca gggccgagct ttggctcccc agcacagggg ctcatgctcc cctccctgga
     4921 ctggggtgag tggtcagtct ctgcccatcg ccactagggc cagagctgtt ggctaagctg
     4981 gggcagggcc taggcctgag cgcctccccc gtctgctccc cagggcggca aggagtacct
     5041 gatgcgggcc cacttcggcc tgcctagtgt ggaggccgaa gacaaggagg gcaagccccc
     5101 gatcagtgtc aagttcgaga tcccttactt cactacctcc ggcatccagg tacgtaaggc
     5161 ccaggcggcc ggggcccaga acagggacag aggccgcagg tgacagatat caatgcagcc
     5221 agcttgggaa agggtgtcag tcagctgcaa tcaggaccct gctgtgctgt gggcctctgc
     5281 ctccccacag acagggtgag gccaggctgc ctctgctcag ccctgacccc tgagctcaaa
     5341 gctcactcat tctgccccaa actagcattt gaggctctgc cgtccactca tgccccatgg
     5401 gacctctttt ccctggcacc ccctcaaaag cccacagagg gaggagaggc tcccagagag
     5461 ccaggggcct tctggccggg ccccccaagc aggctgctga ggccgtgtct gcaagccaca
     5521 tcctggctgc tcaccacttc cagaggcggc tctgccctcc cacacatttg catggtccag
     5581 acaaatccct ttcacccccc tgaggcctgt gctcatcctg tcgtcatggc ctcccctgcc
     5641 agccccaggc tgccccagca ggaaagatta aggggggacc aacagggcgg gagcctgcgg
     5701 tgctcagggc cctgctacct gtgccccagg tgcgctacct gaagatcatt gagaagagtg
     5761 ggtaccaggc cctgccctgg gtgcgttata tcacgcagaa tggaggtgag tgaggcccta
     5821 cccgtggggt ggggttgtac tgaggggtcc tctgtggctg cccccagggt ggagccatcg
     5881 ggtcgggtcc cgaaagcagg gagggtgcag agcccagcat gacgcctccc tcctgtctcc
     5941 tagattacca gctccggacc cagggatcag ggtcaggctc tggttccggc tcaggatcca
     6001 tggcagaaat cggtactggc tttccattcg acccccatta tgtggaagtc ctgggcgagc
     6061 gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta
     6121 acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg acccatcgct
     6181 gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg ggttatttct
     6241 tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg gaagaggtcg
     6301 tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag cgcaatccag
     6361 agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc tgggacgaat
     6421 ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc ggccgcaagc
     6481 tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc
     6541 tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt gaccgcgagc
     6601 cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc
     6661 tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg ctgttctggg
     6721 gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa agcctgccta
     6781 actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac aacccggacc
     6841 tgatcggcag cgagatcgcg cgctggctgt ccacgctcga gatttccggc ccaagcagac
     6901 tggaggagga gctgagaaga agactgaccg agccatgagc tagcccgcga ctctagatca
     6961 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccac
//
