LOCUS       sequence-457076-        6239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology repair plasmid for endogenous tagging of Rab11 at the 
            N-terminus with Halotag and 3x 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 6239)
  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 6239)
  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: loxP-G418-loxP-3xALFA-Halo-Rab11 HR
FEATURES             Location/Qualifiers
     source          1..6239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(117..141)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(241..1032,1033..1101))
                     /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     864..883
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1102..1206)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1305..1327
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1319..1336
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1320..1336
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2103..2136)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2138..2495
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2138..2329
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2161..2181)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2346..2481
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2530..3324
                     /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             2530..3324
                     /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(2584..2603)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3194..3213
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3512..3531)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     protein_bind    complement(3579..3612)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3642..3680
                     /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"
     CDS             3684..3722
                     /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"
     CDS             3726..3764
                     /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"
     CDS             3774..4664
                     /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             complement(5391..5408)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(5463..5479)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5463..5479)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5476..5498)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(5651..6239)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5731..5750)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctctat tcaacaaagc cgccgtcccg
       61 tcaagtcagc gtaaatgggt agggggcttc aaatcgtcct cgtgatacca attcggagcc
      121 tgcttttttg tacaaacttg ttgataatgg caattcaagg atcttcacct agatcctttt
      181 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
      241 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
      301 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      361 cagtgctgca atgataccgc gagagccacg ctcaccggct ccagatttat cagcaataaa
      421 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      481 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      541 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      601 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      661 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      721 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      781 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      841 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      901 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      961 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     1021 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     1081 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     1141 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     1201 tccgcgcaca tttccccgaa aagtgccaga tacctgaaac aaaacccatc gtacggccaa
     1261 ggaagtctcc aataactgtg atccaccaca agcgccaggg ttttcccagt cacgacgttg
     1321 taaaacgacg gccagtcatg cataatccgc acgcatctgg aataaggaag tgccattccg
     1381 cctgacctgc ggccgccccc ccgccaggta gcagatagca cctgaaatgc agtgggaaat
     1441 ttgtgaggtc ttcataggca aggattaaat tttacatctc catcttaaga tgtgtcatag
     1501 ttgttcaatt acttagtgac caaaaaagtg aattaaagat gttgtcaaga ttaaaaaaac
     1561 aaaaaccaat gcaggttcca ccgagatttg aactcggatc gctggattca gagtccagag
     1621 tgctaaccat tacaccatgg aaccgccacg catgtgtagc tgccttcggc tgtctaatcc
     1681 tcagagaacc ccgcccccat ccacaaaccc accactcaca ggcggtcccg cctggttcca
     1741 gcgagccgct tccggcacgg tagctcgaga aatgagcaag cggccactaa gactatggta
     1801 gctaggagtt ccaggactca gtttcccctt tgagcctcct ttagcgacta aagcttgaag
     1861 ccccacgcat ctcgactctc gcgcacaccg cccttgttgg gctcaggggc ggggcgccgc
     1921 ccccggaagt acttcccctt aaaggctggg gcctgccgga aatggcgcag cggcagggag
     1981 gggctcttca cccagtccgg cagttgaagc tcggcgctcg ggttacccct gcagcgacgc
     2041 cccctggtcc cacagatacc actgctgctc ccgccctttc gctcctcggc agcgcagcta
     2101 gcataacttc gtatagcata cattatacga agttatcctg aggcggaaag aaccagctgt
     2161 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2221 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     2281 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     2341 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     2401 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     2461 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     2521 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     2581 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     2641 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     2701 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     2761 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     2821 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     2881 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     2941 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     3001 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     3061 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     3121 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     3181 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     3241 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     3301 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     3361 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     3421 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     3481 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     3541 gaacccgcgc tatgacggca ataaaaagac agaataaaat aacttcgtat agcatacatt
     3601 atacgaagtt atctcgaggt atcatctaac ggatcggtat gagcagactg gaggaggagc
     3661 tgagaagaag actgaccgag ggcagccgtc tggaagaaga actgcgtcgt cgtctgaccg
     3721 aaggctctcg tttagaagaa gaattacgtc gtcgtttaac tgaaggctcc ggaatggcag
     3781 aaatcggtac tggctttcca ttcgaccccc attatgtgga agtcctgggc gagcgcatgc
     3841 actacgtcga tgttggtccg cgcgatggca cccctgtgct gttcctgcac ggtaacccga
     3901 cctcctccta cgtgtggcgc aacatcatcc cgcatgttgc accgacccat cgctgcattg
     3961 ctccagacct gatcggtatg ggcaaatccg acaaaccaga cctgggttat ttcttcgacg
     4021 accacgtccg cttcatggat gccttcatcg aagccctggg tctggaagag gtcgtcctgg
     4081 tcattcacga ctggggctcc gctctgggtt tccactgggc caagcgcaat ccagagcgcg
     4141 tcaaaggtat tgcatttatg gagttcatcc gccctatccc gacctgggac gaatggccag
     4201 aatttgcccg cgagaccttc caggccttcc gcaccaccga cgtcggccgc aagctgatca
     4261 tcgatcagaa cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg
     4321 aagtcgagat ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt
     4381 ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg
     4441 aagaatacat ggactggctg caccagtccc ctgtcccgaa gctgctgttc tggggcaccc
     4501 caggcgttct gatcccaccg gccgaagccg ctcgcctggc caaaagcctg cctaactgca
     4561 aggctgtgga catcggcccg ggtctgaatc tgctgcaaga agacaacccg gacctgatcg
     4621 gcagcgagat cgcgcgctgg ctgtcgacgc tcgagatttc cggcggatcc ggatcagggt
     4681 caggctctgg ttccggctca atgggcaccc gcgacgacga atatgactac ctctttaaag
     4741 gtgaggccat gggctctcgc actctacaca gtcctcgttc ggggacccgg gccactcccg
     4801 gtggaccctc gtgccggcca cccctgcact gatataggcc tccctcagcc cttccttttt
     4861 gtgcggttcc gtctcctacc cagctcagcc tcttctcccc cgctcagaca ggggtcccca
     4921 tcacatgccg ctctctgagc gacctctcca taggccttcg ctggcctcag agcccctccc
     4981 tgcgtgtcct tcccctggcg gactgccttc tcccacatcg tcgaattcct ttccccgggt
     5041 tctacggccc cgccgctcct cccaccatct ctcttttcgg gtgtagcgcc ccctccccct
     5101 cggcgtacac ccttcccagc tcgcgtcctc tcccgaagcc cctctgacgg gttcttcgct
     5161 tccctcttgg ccttgccttc ggtgcagact cccattacag gtctttttct tatctgccct
     5221 ctttcccccc tcgagcgagc gtctcttctg tatctcccct ccacttggac ggccgcccct
     5281 ctaagcttct cccatcccgg gtccaggacc ccgtaaaatc tgagcgggcc aagggcctcc
     5341 gcccccaccc tccgccccgc ggttctcgcg gtccttaacc ttctctgacc gctccctctc
     5401 ggtaccaggc taggtggagg ctcagtgatg ataagtctgc gatggtggat gcatgtgtca
     5461 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca gagggcacaa tcctattccg
     5521 cgctatccga caatctccaa gacattaggt ggagttcagt tcggcgtatg gcatatgtcg
     5581 ctggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5641 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5701 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5761 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5821 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5881 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5941 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6001 cagctactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6061 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6121 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6181 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaa
//
