LOCUS       sequence-452254-        4990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate assembly of donor plasmids to knock-in mScarlet3 
            (BsmBI) using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4990)
  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 4990)
  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-mScarlet3
FEATURES             Location/Qualifiers
     source          1..4990
                     /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      1552..1561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1558..1560,1561..1563,1564..2277)
                     /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(1558..1560,1561..1563,1564..2274)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1603..1624)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1864..1883)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2211..2232
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2404..2525
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2441..2460)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2495..2514
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3218..3237)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3337..3359
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3397..3415)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3483..3587
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3588..3656,3657..4448)
                     /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(3806..3825)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4619..4990,1..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 ctctatggat agcaccgagg cagtgatcaa ggagttcatg cggttcaagg
      721 tgcacatgga gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc
      781 gcccctacga gggcacccag accgccaagc tgagggtgac caagggtggc cccctgccct
      841 tctcctggga catcctgtcc cctcagttca tgtacggctc cagggccttc acgaagcacc
      901 ccgccgacat ccccgactac tggaagcagt ccttccccga gggcttcaag tgggagcgcg
      961 tgatgaactt cgaggacggc ggcgccgtgt ccgtggccca ggacacctcc ctggaggacg
     1021 gcaccctgat ctacaaggtg aagctccgcg gcaccaactt ccctcctgac ggccccgtaa
     1081 tgcagaagaa gacaatgggc tgggaagcat ccaccgagcg gttgtacccc gaggacgtcg
     1141 tgctgaaggg cgacattaag atggccctgc gcctgaagga cggcggccgc tacctggcgg
     1201 acttcaagta acctaggtag ccgcgttgtt atgatcgtac tacgctagcg tagtacgatc
     1261 ataacaacgc gggtagtacg atcataacaa cgcggggatc taattcaatt agagactaat
     1321 tcaattagag ctaattcaat taggatccaa gcttatcgat ttcgaaccct cgaccgccgg
     1381 agtataaata gaggcgcttc gtctacggag cgacaattca attcaaacaa gcaaagtgaa
     1441 cacgtcgcta agcgaaagct aagcaaataa acaagcgcag ctgaacaagc taaacaatcg
     1501 gggtaccgct agagtcgacg gtaccgcggg cccgggatcc accggccggt cgccaccatg
     1561 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1621 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1681 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1741 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1801 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1861 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1921 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1981 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2041 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2101 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2161 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2221 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc
     2281 ggccggccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     2341 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     2401 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2461 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2521 tcttaccgct ggtactgtat ggatgtaccc gctggtactg tatggatgta cgtcgacgta
     2581 catccataca gtaccagcgg actagtgctg agggtgacca agggtggccc cctgcccttc
     2641 tcctgggaca tcctgtcccc tcagttcatg tacggctcca gggccttcac gaagcacccc
     2701 gccgacatcc ccgactactg gaagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg
     2761 atgaacttcg aggacggcgg cgccgtgtcc gtggcccagg acacctccct ggaggacggc
     2821 accctgatct acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg
     2881 cagaagaaga caatgggctg ggaagcatcc accgagcggt tgtaccccga ggacgtcgtg
     2941 ctgaagggcg acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac
     3001 ttcaagacca cctacagggc caagaagccc gtgcagatgc ccggcgcctt caacatcgac
     3061 cgcaagttgg acatcacatc ccacaacgag gactacaccg tggtggaaca gtacgaacgc
     3121 tccgtggccc gccactccac cggcggctcc ggtggctcct ccggcgagac ggctcgaggg
     3181 catgccttaa gagatctggt acctggtgca ctctcagtac aatctgctct gatgccgcat
     3241 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3301 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3361 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     3421 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     3481 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3541 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3601 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3661 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     3721 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3781 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     3841 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     3901 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3961 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4021 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4081 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4141 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4201 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4261 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     4321 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     4381 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4441 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4501 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4561 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4621 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4681 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4741 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4801 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4861 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4921 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4981 acaccgaact
//
