LOCUS       sequence-456982-        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"
     primer_bind     complement(64..83)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     674..693
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(782..1139)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      796..931
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        948..1139
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     1096..1116
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     protein_bind    1141..1174
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1941..1958)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1941..1957)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1950..1972)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        2071..2175
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2176..2244,2245..3036)
                     /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     complement(2394..2413)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    3136..3160
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     rep_origin      3277..3865
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3766..3785
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4018..4040
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4037..4053
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4037..4053
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4108..4125
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(4852..5742)
                     /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(5752..5790)
                     /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             complement(5794..5832)
                     /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             complement(5836..5874)
                     /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"
     protein_bind    5904..5937
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     5985..6004
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(join(6192..6239,1..747))
                     /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             complement(join(6192..6239,1..747))
                     /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"
ORIGIN
        1 gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc
       61 agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc
      121 acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc
      181 gccatgggtc acgacgagat cctcgccgtc gggcatgctc gccttgagcc tggcgaacag
      241 ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc
      301 ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt
      361 agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc
      421 aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc
      481 ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag
      541 ccacgatagc cgcgctgcct cgtcttgcag ttcattcagg gcaccggaca ggtcggtctt
      601 gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc
      661 gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc
      721 tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcgat
      781 ctttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg
      841 ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat
      901 gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg
      961 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     1021 cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     1081 ctggggactt tccacaccct aactgacaca cattccacag ctggttcttt ccgcctcagg
     1141 ataacttcgt ataatgtatg ctatacgaag ttatgctagc tgcgctgccg aggagcgaaa
     1201 gggcgggagc agcagtggta tctgtgggac cagggggcgt cgctgcaggg gtaacccgag
     1261 cgccgagctt caactgccgg actgggtgaa gagcccctcc ctgccgctgc gccatttccg
     1321 gcaggcccca gcctttaagg ggaagtactt ccgggggcgg cgccccgccc ctgagcccaa
     1381 caagggcggt gtgcgcgaga gtcgagatgc gtggggcttc aagctttagt cgctaaagga
     1441 ggctcaaagg ggaaactgag tcctggaact cctagctacc atagtcttag tggccgcttg
     1501 ctcatttctc gagctaccgt gccggaagcg gctcgctgga accaggcggg accgcctgtg
     1561 agtggtgggt ttgtggatgg gggcggggtt ctctgaggat tagacagccg aaggcagcta
     1621 cacatgcgtg gcggttccat ggtgtaatgg ttagcactct ggactctgaa tccagcgatc
     1681 cgagttcaaa tctcggtgga acctgcattg gtttttgttt ttttaatctt gacaacatct
     1741 ttaattcact tttttggtca ctaagtaatt gaacaactat gacacatctt aagatggaga
     1801 tgtaaaattt aatccttgcc tatgaagacc tcacaaattt cccactgcat ttcaggtgct
     1861 atctgctacc tggcgggggg gcggccgcag gtcaggcgga atggcacttc cttattccag
     1921 atgcgtgcgg attatgcatg actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     1981 ggcgcttgtg gtggatcaca gttattggag acttccttgg ccgtacgatg ggttttgttt
     2041 caggtatctg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2101 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2161 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2221 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2281 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2341 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2401 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2461 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2521 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2581 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2641 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2701 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2761 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2821 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2881 ggtgagcgtg gctctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2941 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3001 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3061 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3121 gaattgccat tatcaacaag tttgtacaaa aaagcaggct ccgaattggt atcacgagga
     3181 cgatttgaag ccccctaccc atttacgctg acttgacggg acggcggctt tgttgaatag
     3241 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3301 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3361 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3421 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3481 atacctcgct ctgctaatcc tgttaccagt agctgctgcc agtggcgata agtcgtgtct
     3541 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3601 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3661 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3721 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3781 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3841 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3901 cttttgctgg ccttttgctc acatgttctt tccagcgaca tatgccatac gccgaactga
     3961 actccaccta atgtcttgga gattgtcgga tagcgcggaa taggattgtg ccctctgagc
     4021 ggataacaat ttcacacagg aaacagctat gaccatgaca catgcatcca ccatcgcaga
     4081 cttatcatca ctgagcctcc acctagcctg gtaccgagag ggagcggtca gagaaggtta
     4141 aggaccgcga gaaccgcggg gcggagggtg ggggcggagg cccttggccc gctcagattt
     4201 tacggggtcc tggacccggg atgggagaag cttagagggg cggccgtcca agtggagggg
     4261 agatacagaa gagacgctcg ctcgaggggg gaaagagggc agataagaaa aagacctgta
     4321 atgggagtct gcaccgaagg caaggccaag agggaagcga agaacccgtc agaggggctt
     4381 cgggagagga cgcgagctgg gaagggtgta cgccgagggg gagggggcgc tacacccgaa
     4441 aagagagatg gtgggaggag cggcggggcc gtagaacccg gggaaaggaa ttcgacgatg
     4501 tgggagaagg cagtccgcca ggggaaggac acgcagggag gggctctgag gccagcgaag
     4561 gcctatggag aggtcgctca gagagcggca tgtgatgggg acccctgtct gagcggggga
     4621 gaagaggctg agctgggtag gagacggaac cgcacaaaaa ggaagggctg agggaggcct
     4681 atatcagtgc aggggtggcc ggcacgaggg tccaccggga gtggcccggg tccccgaacg
     4741 aggactgtgt agagtgcgag agcccatggc ctcaccttta aagaggtagt catattcgtc
     4801 gtcgcgggtg cccattgagc cggaaccaga gcctgaccct gatccggatc cgccggaaat
     4861 ctcgagcgtc gacagccagc gcgcgatctc gctgccgatc aggtccgggt tgtcttcttg
     4921 cagcagattc agacccgggc cgatgtccac agccttgcag ttaggcaggc ttttggccag
     4981 gcgagcggct tcggccggtg ggatcagaac gcctggggtg ccccagaaca gcagcttcgg
     5041 gacaggggac tggtgcagcc agtccatgta ttcttcgacc agcgcgacga tgttcgctgg
     5101 ctcaccggcg attggcagct cgtttgggaa gcgccacagt ggctcgcggt caacaggatt
     5161 caggaacggc tcgcggtaat ggtccatctc gacttcagtc agcgggcgga cgacacccat
     5221 cggcagcgta ccctcgataa aaacgttctg atcgatgatc agcttgcggc cgacgtcggt
     5281 ggtgcggaag gcctggaagg tctcgcgggc aaattctggc cattcgtccc aggtcgggat
     5341 agggcggatg aactccataa atgcaatacc tttgacgcgc tctggattgc gcttggccca
     5401 gtggaaaccc agagcggagc cccagtcgtg aatgaccagg acgacctctt ccagacccag
     5461 ggcttcgatg aaggcatcca tgaagcggac gtggtcgtcg aagaaataac ccaggtctgg
     5521 tttgtcggat ttgcccatac cgatcaggtc tggagcaatg cagcgatggg tcggtgcaac
     5581 atgcgggatg atgttgcgcc acacgtagga ggaggtcggg ttaccgtgca ggaacagcac
     5641 aggggtgcca tcgcgcggac caacatcgac gtagtgcatg cgctcgccca ggacttccac
     5701 ataatggggg tcgaatggaa agccagtacc gatttctgcc attccggagc cttcagttaa
     5761 acgacgacgt aattcttctt ctaaacgaga gccttcggtc agacgacgac gcagttcttc
     5821 ttccagacgg ctgccctcgg tcagtcttct tctcagctcc tcctccagtc tgctcatacc
     5881 gatccgttag atgatacctc gagataactt cgtataatgt atgctatacg aagttatttt
     5941 attctgtctt tttattgccg tcatagcgcg ggttccttcc ggtattgtct ccttccgtgt
     6001 ttcagttagc ctccccctag ggtgggcgaa gaactccagc atgagatccc cgcgctggag
     6061 gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc
     6121 ggtggaatcg aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc
     6181 cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatc
//
