LOCUS       sequence-449819-        5364 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate assembly (BsmBI) of donor plasmids to knock-in 
            EYFP and HA tag using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5364)
  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 5364)
  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-EYFP:HA
FEATURES             Location/Qualifiers
     source          1..5364
                     /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     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     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             join(670..672,673..675,676..1188)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(715..736)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1493..1502
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1499..2176
                     /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(1629..1647)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1679..1699)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1897..1916
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2091..2114
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2297..2418
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2334..2353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2388..2407
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             2850..3101
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     3038..3059
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3120..3146
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(3179..3197)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3218..3234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3218..3234)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3231..3253)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3242..3258
                     /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(3266..3272,3273..3290,3291..3296))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3311..3332
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3449..3466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3620..4208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3700..3719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4379..5170,5171..5239))
                     /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     5002..5021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5240..5344)
                     /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 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgt
      661 ctcggctcta tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      721 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
      781 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      841 tggcccaccc tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg ctaccccgac
      901 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
      961 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1021 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1081 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1141 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgaggc ctaggtagcc
     1201 gcgttgttat gatcgtacta cgctagcgta gtacgatcat aacaacgcgg gtagtacgat
     1261 cataacaacg cggggatcta attcaattag agactaattc aattagagct aattcaatta
     1321 ggatccaagc ttatcgattt cgaaccctcg accgccggag tataaataga ggcgcttcgt
     1381 ctacggagcg acaattcaat tcaaacaagc aaagtgaaca cgtcgctaag cgaaagctaa
     1441 gcaaataaac aagcgcagct gaacaagcta aacaatcggc tcgaagccgg tcgccaccat
     1501 ggcctcctcc gaggacgtca tcaaggagtt catgcgcttc aaggtgcgca tggagggctc
     1561 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     1621 ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct gggacatcct
     1681 gtccccccag ttccagtacg gctccaaggt gtacgtgaag caccccgccg acatccccga
     1741 ctacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1801 cggcggcgtg gtgaccgtga cccaggactc ctccctccag gacggctcct tcatctacaa
     1861 ggtgaagttc atcggcgtga acttcccctc cgacggcccc gtaatgcaga agaagactat
     1921 gggctgggag gcgtccaccg agcgcctgta cccccgcgac ggcgtgctga agggcgagat
     1981 ccacaaggcc ctgaagctga aggacggcgg ccactacctg gtggagttca agtccatcta
     2041 catggccaag aagcccgtgc agctgcccgg ctactactac gtggactcca agctggacat
     2101 cacctcccac aacgaggact acaccatcgt ggagcagtac gagcgcgccg agggccgcca
     2161 ccacctgttc ctgtagcggc cgcgactcta gatcataatc agccatacca catttgtaga
     2221 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     2281 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2341 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2401 actcatcaat gtatcttacc gctggtactg tatggatgta cccgctggta ctgtatggat
     2461 gtacgtcgac gtacatccat acagtaccag cggactagtc ctacggcaag ctgaccctga
     2521 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttcg
     2581 gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca
     2641 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     2701 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     2761 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     2821 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     2881 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     2941 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt
     3001 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     3061 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggagggtct gcagggggat
     3121 acccatacga tgttccagat tacgctggat ccggcgagac ggtacccagc ttttgttccc
     3181 tttagtgagg gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3241 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3301 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3361 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3421 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3481 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3541 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3601 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3661 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3721 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3781 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3841 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3901 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3961 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4021 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4081 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4141 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4201 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4261 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4321 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4381 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4441 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4501 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4561 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4621 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4681 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4741 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4801 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4861 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4921 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4981 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5041 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5101 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5161 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5221 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5281 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5341 cgcgcacatt tccccgaaaa gtgc
//
