LOCUS       sequence-449870-        4906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate assembly (BsmBI) of donor plasmids to knock-in 
            Dendra2 using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4906)
  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 4906)
  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-Dendra2
FEATURES             Location/Qualifiers
     source          1..4906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(541..546,547..564,565..571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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     584..606
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     603..619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     regulatory      1488..1497
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1494..2171
                     /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(1624..1642)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1674..1694)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2086..2109
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2292..2413
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2329..2348)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2383..2402
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3134..3153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3253..3275
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3313..3331)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3399..3503
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3504..3572,3573..4364)
                     /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     complement(3722..3741)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4535..4906,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacaggaaac agctatgacc atgattacgc cagagctcac gcgtaccggt ctcgaggaat
      661 tccgtctcgg ctctatgaac accccgggaa ttaacctgat caaggaggac atgcgcgtga
      721 aggtgcacat ggagggcaac gtgaacggcc acgccttcgt gatcgagggc gagggcaagg
      781 gcaagaccta cgagggcacc cagaccgcca acctgaccgt gaaggagggc gcccccctgc
      841 ccttcagcta cgacatcctg accaccgccg tgcactacgg caaccgggtg ttcaccaagt
      901 accccgagga catccccgac tacttcaagc agagcttccc cgagggctac agctgggagc
      961 gcaccatgac cttcgaggac aagggcatct gcaccatccg cagcgacatc agcctggagg
     1021 gcgactgctt cttccagaac gtgcgcttca agggcaccaa cttccccccc aacggccccg
     1081 tgatgcagaa gaagaccctg aagtgggagc ccagcaccga gaagctgcac gtgcgcgacg
     1141 gcctgctggt gggcaacatc aacatggccc tgctgctgga gggccctagg tagccgcgtt
     1201 gttatgatcg tactacgcta gcgtagtacg atcataacaa cgcgggtagt acgatcataa
     1261 caacgcgggg atctaattca attagagact aattcaatta gagctaattc aattaggatc
     1321 caagcttatc gatttcgaac cctcgaccgc cggagtataa atagaggcgc ttcgtctacg
     1381 gagcgacaat tcaattcaaa caagcaaagt gaacacgtcg ctaagcgaaa gctaagcaaa
     1441 taaacaagcg cagctgaaca agctaaacaa tcggctcgaa gccggtcgcc accatggcct
     1501 cctccgagga cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag ggctccgtga
     1561 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     1621 ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
     1681 cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca
     1741 agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     1801 gcgtggtgac cgtgacccag gactcctccc tccaggacgg ctccttcatc tacaaggtga
     1861 agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaag actatgggct
     1921 gggaggcgtc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca
     1981 aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg
     2041 ccaagaagcc cgtgcagctg cccggctact actacgtgga ctccaagctg gacatcacct
     2101 cccacaacga ggactacacc atcgtggagc agtacgagcg cgccgagggc cgccaccacc
     2161 tgttcctgta gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt
     2221 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2281 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2341 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2401 tcaatgtatc ttaccgctgg tactgtatgg atgtacccgc tggtactgta tggatgtacg
     2461 tcgacgtaca tccatacagt accagcggac tagtgcccta cgagggcacc cagaccgcca
     2521 acctgaccgt gaaggagggc gcccccctgc ccttcagcta cgacatcctg accaccgccg
     2581 tgcactacgg caaccgggtg ttcaccaagt accccgagga catccccgac tacttcaagc
     2641 agagcttccc cgagggctac agctgggagc gcaccatgac cttcgaggac aagggcatct
     2701 gcaccatccg cagcgacatc agcctggagg gcgactgctt cttccagaac gtgcgcttca
     2761 agggcaccaa cttccccccc aacggccccg tgatgcagaa gaagaccctg aagtgggagc
     2821 ccagcaccga gaagctgcac gtgcgcgacg gcctgctggt gggcaacatc aacatggccc
     2881 tgctgctgga gggcggcggc cactacctgt gcgacttcaa gaccacctac aaggccaaga
     2941 aggtggtgca gctgcccgac gcccacttcg tggaccaccg catcgagatc ctgggcaacg
     3001 acagcgacta caacaaggtg aagctgtacg agcacgccgt ggcccgctac agccccctgc
     3061 ccagccaggt gtggtccggc gagacgtccg gcgccgcatg ccttaagaga tctggtacct
     3121 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     3181 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3241 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3301 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3361 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3421 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3481 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3541 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3601 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3661 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3721 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3781 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3841 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3901 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3961 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4021 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4081 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4141 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4201 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4261 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4321 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4381 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4441 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4501 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4561 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4621 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4681 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4741 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4801 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4861 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaact
//
