LOCUS       sequence-452256-        3243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate assembly (BsmBI) of donor plasmids to knock-in 
            mScarlet3:ALFA using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3243)
  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 3243)
  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:ALFA
FEATURES             Location/Qualifiers
     source          1..3243
                     /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"
     CDS             675..1361
                     /codon_start=1
                     /product="bright cysteine-free monomeric red fluorescent 
                     protein with fast and complete maturation (Gadella et al., 
                     2023)"
                     /label=mScarlet3
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LRVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGA
                     VSVAQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKM
                     ALRLKDGGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHS
                     TGGSGGS"
     primer_bind     1073..1092
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1371..1409
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     primer_bind     complement(1471..1490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1590..1612
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1650..1668)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1736..1840
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1841..1909,1910..2701)
                     /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(2059..2078)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2872..3243,1..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 acttcaagac cacctacagg gccaagaagc ccgtgcagat gcccggcgcc ttcaacatcg
     1261 accgcaagtt ggacatcaca tcccacaacg aggactacac cgtggtggaa cagtacgaac
     1321 gctccgtggc ccgccactcc accggcggct ccggtggctc cggatctcca tcacgtttgg
     1381 aagaggaact gaggcgccgc ttaactgaac cttccggcga gacgactcga gggcatgcct
     1441 taagagatct ggtacctggt gcactctcag tacaatctgc tctgatgccg catagttaag
     1501 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     1561 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     1621 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     1681 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     1741 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1801 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1861 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1921 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1981 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     2041 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     2101 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     2161 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2221 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2281 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2341 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     2401 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2461 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2521 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2581 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2641 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2701 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2761 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2821 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2881 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2941 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3001 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3061 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3121 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3181 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3241 act
//
