LOCUS       sequence-480339-        2754 bp    DNA     circular SYN 15-DEC-2025
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 Dec 15, 2025 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"
     primer_bind     152..169
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    286..307
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(322..327,328..345,346..352)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    360..376
                     /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     365..387
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     384..400
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     384..400
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(448..474)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(508..546)
                     /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(583..600)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(583..599)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(592..614)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(808..827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     927..949
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(987..1005)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1073..1177
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1178..1993
                     /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(1251..1270)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2164..2752
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2653..2672
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
       61 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      121 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaca
      181 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      241 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      301 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      361 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgctcgag ccaagctcgg
      421 cgcgcccgtc tcgccggaac cggatccagc gtaatctgga acatcgtatg ggtaggagcc
      481 gcccgaacct gatcctccag acccaggttc agttaagcgg cgtctgagtt cctcttccaa
      541 acgtgatggt ccagagccag agccgagacg acgcgtcaat tcactggccg tcgttttaca
      601 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
      661 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
      721 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
      781 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
      841 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
      901 cgcttacaga caagctgtga cagtctccgg gagctgcatg tgtcagaggt tttcaccgtc
      961 atcaccgaaa cgcgcgagac aaaagggcct cgtgatacgc ctatttttat aggttaatgt
     1021 catgataata atggtttctt agacgtcagg tggcactttt cgggaaaatg tgcgcggaac
     1081 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1141 ctgataaatg cttcaataat attgaaaaag gaagagtatg agccatattc aacgggaaac
     1201 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     1261 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     1321 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     1381 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     1441 ccgtactcct gatgatgcat ggttactcac cactgcgatc cctgggaaaa cagcattcca
     1501 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     1561 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     1621 tttcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     1681 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     1741 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     1801 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     1861 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     1921 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     1981 tgagtttttc taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2041 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2101 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2161 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2221 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2281 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2341 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2401 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2461 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2521 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2581 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2641 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2701 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaa
//
