LOCUS       sequence-465053-        5006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate (BsmBI) assembly of donor plasmids to knock-in 
            mCherry using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5006)
  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 5006)
  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-mCherry
FEATURES             Location/Qualifiers
     source          1..5006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     141..158
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    275..296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(311..316,317..334,335..341)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    349..365
                     /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     354..376
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     373..389
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     373..389
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     regulatory      1316..1325
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1322..1324,1325..1327,1328..2041)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1322..1324,1325..1327,1328..2038)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1367..1388)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1628..1647)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1975..1996
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2168..2289
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2205..2224)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2259..2278
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3004..3023)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3123..3145
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3183..3201)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3269..3373
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3374..3442,3443..4234)
                     /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(3592..3611)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4405..4993
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4894..4913
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
       61 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
      121 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
      181 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
      241 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      301 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      361 aacaatttca cacaggaaac agctatgacc atgattacgc cagagctcac gcgtaccggt
      421 ctcgaggaat tcgccggctc tatggtgagc aagggcgagg aggataacat ggccatcatc
      481 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
      541 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
      601 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
      661 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
      721 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
      781 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
      841 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
      901 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
      961 gacggcggct ccggccctag gtagccgcgt tgttatgatc gtactacgct agcgtagtac
     1021 gatcataaca acgcgggtag tacgatcata acaacgcggg gatctaattc aattagagac
     1081 taattcaatt agagctaatt caattaggat ccaagcttat cgatttcgaa ccctcgaccg
     1141 ccggagtata aatagaggcg cttcgtctac ggagcgacaa ttcaattcaa acaagcaaag
     1201 tgaacacgtc gctaagcgaa agctaagcaa ataaacaagc gcagctgaac aagctaaaca
     1261 atcggggtac cgctagagtc gacggtaccg cgggcccggg atccaccggc cggtcgccac
     1321 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1381 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1441 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1501 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1561 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1621 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1681 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1741 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1801 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1861 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1921 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1981 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     2041 aagcggccgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     2101 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2161 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2221 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2281 tgtatcttac cgctggtact gtatggatgt acccgctggt actgtatgga tgtacgtcga
     2341 cgtacatcca tacagtacca gcggactagt ggctctcgag ggcacccaga ccgccaagct
     2401 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     2461 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     2521 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     2581 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     2641 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     2701 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     2761 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     2821 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     2881 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     2941 ggacgagctg tacaagtccg gcgccgcatg ccttaagaga tctggtacct ggtgcactct
     3001 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     3061 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3121 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     3181 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     3241 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3301 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3361 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3421 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3481 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3541 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3601 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3661 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3721 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3781 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3841 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3901 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3961 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4021 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     4081 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4141 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     4201 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4261 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4321 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4381 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4441 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4501 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4561 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4621 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4681 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4741 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4801 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4861 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4921 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4981 gagcctatgg aaaaacgcca gcaacg
//
