LOCUS       sequence-503079-        2754 bp    DNA     circular SYN 17-MAR-2026
DEFINITION  Permits GoldenGate assembly (BsmBI) of donor plasmids to knock-in 
            ALFA and HA tags using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2754)
  AUTHORS   Aguilar G, Bauer M, Vigano MA, Schnider ST, Brugger L, 
            Jimenez-Jimenez C, Guerrero I, Affolter M
  TITLE     Seamless knockins in Drosophila via CRISPR-triggered single-strand 
            annealing.
  JOURNAL   Dev Cell. 2024 Oct 7;59(19):2672-2686.e5. doi: 
            10.1016/j.devcel.2024.06.004. Epub 2024 Jul 5.
  PUBMED    38971155
REFERENCE   2  (bases 1 to 2754)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 17, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.devcel.2024.06.004"; journalName: "Dev Cell"; date: 
            "2024-10-7- 7"; volume: "59"; issue: "19"; pages: "2672-2686.e5"
COMMENT     Sequence Label: pSEED-ALFA:HA
FEATURES             Location/Qualifiers
     source          1..2754
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(1039..1065)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1099..1137)
                     /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"
     primer_bind     complement(1174..1191)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1174..1190)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1183..1205)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1399..1418)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1518..1540
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1578..1596)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1664..1768
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1769..2584
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRFAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1842..1861)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaac agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgctcga gccaagctcg gcgcgcccgt
     1021 ctcgccggaa ccggatccag cgtaatctgg aacatcgtat gggtaggagc cgcccgaacc
     1081 tgatcctcca gacccaggtt cagttaagcg gcgtctgagt tcctcttcca aacgtgatgg
     1141 tccagagcca gagccgagac gacgcgtcaa ttcactggcc gtcgttttac aacgtcgtga
     1201 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     1261 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     1321 tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     1381 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     1441 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     1501 acaagctgtg acagtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     1561 acgcgcgaga caaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     1621 aatggtttct tagacgtcag gtggcacttt tcgggaaaat gtgcgcggaa cccctatttg
     1681 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     1741 gcttcaataa tattgaaaaa ggaagagtat gagccatatt caacgggaaa cgtcttgctc
     1801 taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     1861 taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga
     1921 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     1981 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     2041 tgatgatgca tggttactca ccactgcgat ccctgggaaa acagcattcc aggtattaga
     2101 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     2161 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtttcgctca
     2221 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     2281 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     2341 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     2401 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     2461 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     2521 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     2581 ctaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2641 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2701 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
