LOCUS       sequence-452279-        4344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate assembly (BsmBI) of donor plasmids to knock-in 
            OLLAS and HA tags using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4344)
  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 4344)
  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-OLLAS:HA
FEATURES             Location/Qualifiers
     source          1..4344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             670..711
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     CDS             712..738
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1048..1057
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1054..1731
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(1184..1202)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1234..1254)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1452..1471
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1646..1669
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1852..1973
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1889..1908)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1943..1962
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             2061..2102
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     CDS             2103..2129
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(2159..2177)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2198..2214)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2198..2214)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2211..2233)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2222..2238
                     /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)."
     promoter        complement(join(2246..2252,2253..2270,2271..2276))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2291..2312
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2429..2446)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2600..3188)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2680..2699)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3359..4150,4151..4219))
                     /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     3982..4001
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4220..4324)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttctaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgt
      661 ctcggctcta gcggcttcgc gaacgagctg ggaccccgcc tgatgggtaa atatccctat
      721 gacgtcccgg actatgcatc cggccctagg tagccgcgtt gttatgatcg tactacgcta
      781 gcgtagtacg atcataacaa cgcgggtagt acgatcataa caacgcgggg atctaattca
      841 attagagact aattcaatta gagctaattc aattaggatc caagcttatc gatttcgaac
      901 cctcgaccgc cggagtataa atagaggcgc ttcgtctacg gagcgacaat tcaattcaaa
      961 caagcaaagt gaacacgtcg ctaagcgaaa gctaagcaaa taaacaagcg cagctgaaca
     1021 agctaaacaa tcggctcgaa gccggtcgcc accatggcct cctccgagga cgtcatcaag
     1081 gagttcatgc gcttcaaggt gcgcatggag ggctccgtga acggccacga gttcgagatc
     1141 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     1201 aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcca gtacggctcc
     1261 aaggtgtacg tgaagcaccc cgccgacatc cccgactaca agaagctgtc cttccccgag
     1321 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     1381 gactcctccc tccaggacgg ctccttcatc tacaaggtga agttcatcgg cgtgaacttc
     1441 ccctccgacg gccccgtaat gcagaagaag actatgggct gggaggcgtc caccgagcgc
     1501 ctgtaccccc gcgacggcgt gctgaagggc gagatccaca aggccctgaa gctgaaggac
     1561 ggcggccact acctggtgga gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg
     1621 cccggctact actacgtgga ctccaagctg gacatcacct cccacaacga ggactacacc
     1681 atcgtggagc agtacgagcg cgccgagggc cgccaccacc tgttcctgta gcggccgcga
     1741 ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     1801 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     1861 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     1921 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaccgctgg
     1981 tactgtatgg atgtacccgc tggtactgta tggatgtacg tcgacgtaca tccatacagt
     2041 accagcggac tagtggctct agcggcttcg cgaacgagct gggaccccgc ctgatgggta
     2101 aatatcccta tgacgtcccg gactatgcat ccggcgagac ggtacccagc ttttgttccc
     2161 tttagtgagg gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2221 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2281 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2341 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2401 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2461 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2521 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2581 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2641 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2701 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2761 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2821 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2881 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2941 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3001 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3061 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3121 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3181 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3241 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3301 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3361 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3421 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3481 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3541 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3601 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3661 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3721 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3781 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3841 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3901 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3961 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4021 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4081 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4141 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4201 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4261 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4321 cgcgcacatt tccccgaaaa gtgc
//
