LOCUS       sequence-457126-        6203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology repair plasmid for endogenous tagging of Rab11 at the 
            N-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 6203)
  AUTHORS   Adarska P, Fox E, Heyza J, Barnaba C, Schmidt J, Bottanelli F
  TITLE     Protocol for fast antibiotic resistance-based gene editing of 
            mammalian cells with CRISPR-Cas9.
  JOURNAL   STAR Protoc. 2025 Aug 4;6(3):103949. doi: 
            10.1016/j.xpro.2025.103949.
  PUBMED    40768339
REFERENCE   2  (bases 1 to 6203)
  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: "STAR 
            Protoc."; date: "2025-08-4"; volume: "6(3):103949. doi"; pages: " 
            10.1016/j.xpro.2025.103949"
COMMENT     Sequence Label: loxP-G418-LoxP-TurboID-Rab11 HR
FEATURES             Location/Qualifiers
     source          1..6203
                     /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"
     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             3624..3665
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3672..4628
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             complement(5355..5372)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(5427..5443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5427..5443)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5440..5462)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(5615..6203)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5695..5714)
                     /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 atcagctagc atgggcaagc ccatccccaa ccccctgctg ggcctggaca
     3661 gcaccgaatt caaagacaat actgtgcctc tgaagctgat cgctctcctg gctaatggcg
     3721 agttccatag tggcgaacag ctgggagaaa ccctgggcat gtccagggcc gctatcaaca
     3781 agcacattca gactctgcgc gactggggcg tggacgtgtt caccgtgccc ggaaagggct
     3841 actctctgcc cgagcctatc ccgctgctga acgctaaaca gattctggga cagctggacg
     3901 gcgggagcgt ggcagtcctg cctgtggtcg actccaccaa tcagtacctg ctggatcgaa
     3961 tcggcgagct gaagagtggg gatgcttgca ttgcagaata tcagcaggca gggagaggaa
     4021 gcagagggag gaaatggttc tctccttttg gagctaacct gtacctgagt atgttttggc
     4081 gcctgaagcg gggaccagca gcaatcggcc tgggcccggt catcggaatt gtcatggcag
     4141 aagcgctgcg aaagctggga gcagacaagg tgcgagtcaa atggcccaat gacctgtatc
     4201 tgcaggatag aaagctggca ggcatcctgg tggagctggc cggaataaca ggcgatgctg
     4261 cacagatcgt cattggcgcc gggattaacg tggctatgag gcgcgtggag gaaagcgtgg
     4321 tcaatcaggg ctggatcaca ctgcaggaag cagggattaa cctggacagg aatactctgg
     4381 ccgctacgct gatccgagag ctgcgggcag ccctggaact gttcgagcag gaaggcctgg
     4441 ctccatatct gccacggtgg gagaagctgg ataacttcat caatagaccc gtgaagctga
     4501 tcattgggga caaagagatt ttcgggatta gccgggggat tgataaacag ggagccctgc
     4561 tgctggaaca ggacggagtt atcaaaccct ggatgggcgg agaaatcagt ctgcggtctg
     4621 ccgaaaaggg atccggatca gggtcaggct ctggttccgg ctcaatgggc acccgcgacg
     4681 acgaatatga ctacctcttt aaaggtgagg ccatgggctc tcgcactcta cacagtcctc
     4741 gttcggggac ccgggccact cccggtggac cctcgtgccg gccacccctg cactgatata
     4801 ggcctccctc agcccttcct ttttgtgcgg ttccgtctcc tacccagctc agcctcttct
     4861 cccccgctca gacaggggtc cccatcacat gccgctctct gagcgacctc tccataggcc
     4921 ttcgctggcc tcagagcccc tccctgcgtg tccttcccct ggcggactgc cttctcccac
     4981 atcgtcgaat tcctttcccc gggttctacg gccccgccgc tcctcccacc atctctcttt
     5041 tcgggtgtag cgccccctcc ccctcggcgt acacccttcc cagctcgcgt cctctcccga
     5101 agcccctctg acgggttctt cgcttccctc ttggccttgc cttcggtgca gactcccatt
     5161 acaggtcttt ttcttatctg ccctctttcc cccctcgagc gagcgtctct tctgtatctc
     5221 ccctccactt ggacggccgc ccctctaagc ttctcccatc ccgggtccag gaccccgtaa
     5281 aatctgagcg ggccaagggc ctccgccccc accctccgcc ccgcggttct cgcggtcctt
     5341 aaccttctct gaccgctccc tctcggtacc aggctaggtg gaggctcagt gatgataagt
     5401 ctgcgatggt ggatgcatgt gtcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5461 ctcagagggc acaatcctat tccgcgctat ccgacaatct ccaagacatt aggtggagtt
     5521 cagttcggcg tatggcatat gtcgctggaa agaacatgtg agcaaaaggc cagcaaaagg
     5581 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5641 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5701 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5761 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5821 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5881 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5941 gacacgactt atcgccactg gcagcagcta ctggtaacag gattagcaga gcgaggtatg
     6001 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6061 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6121 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6181 cgcgcagaaa aaaaggatct caa
//
