LOCUS       sequence-457127-        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"
     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"
     primer_bind     1453..1472
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     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..4009
                     /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             4013..4051
                     /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"
     polyA_signal    4181..4302
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4218..4237)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4272..4291
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     enhancer        4328..4519
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4351..4371)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             4699..5493
                     /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             4699..5493
                     /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(4753..4772)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5363..5382
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5681..5700)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(6827..6843)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6827..6843)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6840..6862)
                     /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 ggcagaaatc ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg
     3181 catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa
     3241 cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg
     3301 cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt
     3361 cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt
     3421 cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga
     3481 gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg
     3541 gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct
     3601 gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct
     3661 gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc
     3721 actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct
     3781 ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg
     3841 caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa
     3901 ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct
     3961 gatcggcagc gagatcgcgc gctggctgtc cacgctcgag atttccggcc caagcagact
     4021 ggaggaggag ctgagaagaa gactgaccga gccatgagct agcccgcgac tctagatcat
     4081 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     4141 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     4201 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4261 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaaggc
     4321 ggccgccctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     4381 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4441 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4501 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgccccatg
     4561 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4621 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca
     4681 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4741 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4801 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4861 ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     4921 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4981 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     5041 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     5101 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     5161 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     5221 aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     5281 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     5341 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     5401 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     5461 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     5521 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     5581 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5641 tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa
     5701 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaagaa ttcggggctg
     5761 tcgcagccaa caccccagcc tcggagctcc tggtggcagc accaggggac acacctgcca
     5821 aacccaccag atggaggggc cctccctggt ctctggccac cctcccagcc tctgcccagg
     5881 gacccctgcc ttccccaggc catctgctct gccgtcgaca ctcgtctcag aagccccttt
     5941 cccagaagag gctggtcttc aagaagtctc gtttctttgc ccctgaagtc agtttcaggg
     6001 gaaggatgtg aaatttttcc gtgtagaggt tacagccttt tatgctgttg agctcccagg
     6061 taccaaaaag cttggccaac gcttgccagc cagccagctg caggtggcat ctgccacgaa
     6121 ggaagcgcca gcctcgccag gccagcaggg gcgtcgtttt gttgccattt tgttgaacgt
     6181 tatgggttta tgggtgttcc tggaacttgt ctttgtgcat tcgttgctgt ttgtgttacc
     6241 ctcactgtcc ccatgtccca cccacgtcct acggcactca ggaagcactt ggtgaggacg
     6301 agccctcacc cttcttgtct tccttcccgg cagcgcccgc agcgggccat ttacacgtcg
     6361 aggctggcac ctggcgcgct cgggggccac tgtagcgtct gcctgctccc tggactcgca
     6421 ggcctcgcct gtggcgcctt cccagggcca gcctgggtca cgagatgctg tcactcagcc
     6481 agatcagtat tgacccacca ggggaggtgg ggtttggtga gagacgccag cctcagactt
     6541 tttcccactg agggtccaga gagcggggcc acgtgtcacc cacgtctgcg cttggtcacc
     6601 cgtcctcccc accctgtgtg tgtttatgtc atagttacat taaattccat tcattgaata
     6661 ctccacagcc ttcctctgat gcagcgtggt cccaactcct cctgcgtgac cacagggctt
     6721 gcctggtttt gtcgccgacc ttccctgtgg ccccacgagg tgtcaccagc aggctaggtg
     6781 gaggctcagt gatgataagt ctgcgatggt ggatgcatgt gtcatggtca tagctgtttc
     6841 ctgtgtgaaa ttgttatccg ctcagagggc acaatcctat tccgcgctat ccgacaatct
     6901 ccaagacatt aggtggagtt cagttcggcg tatggcatat gtcgctggaa agaacatgtg
     6961 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtt
//
