LOCUS       sequence-449682-        4984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate assembly of donor plasmids (BsmBI) to knock-in 
            mScarlet:ALFA using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4984)
  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 4984)
  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-mGreenLantern:ALFA
FEATURES             Location/Qualifiers
     source          1..4984
                     /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"
     primer_bind     complement(720..741)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     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     1892..1911
                     /label=mCherry-F
                     /note="mCherry, forward 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"
     CDS             3120..3158
                     /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(3212..3231)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3331..3353
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3391..3409)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3477..3581
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3582..3650,3651..4442)
                     /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(3800..3819)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4613..4984,1..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 ctctatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      721 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtccgcggc gagggcgagg
      781 gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      841 tgccctggcc caccctcgtg accaccttag gctacggcgt ggcctgcttc gcccgctacc
      901 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      961 agcgcaccat ctctttcaag gacgacggca cctacaagac ccgcgccgag gtgaagttcg
     1021 agggcgacac cctggtgaac cgcatcgtgc tgaagggcat cgacttcaag gaggacggca
     1081 acatcctggg gcacaagctg gagtacaact tcaacagcca caaggtctat atcacggccg
     1141 acaagcagaa gaacggcatc aaggctaact tcaagacccg ctagcctagg 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 tagtcgatgc caccaacggc aagctgaccc
     2521 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct
     2581 taggctacgg cgtggcctgc ttcgcccgct accccgacca catgaagcag cacgacttct
     2641 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctctttc aaggacgacg
     2701 gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     2761 tgctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2821 acttcaacag ccacaaggtc tatatcacgg ccgacaagca gaagaacggc atcaaggcta
     2881 acttcaagac ccgccacaac gttgaggacg gcggcgtgca gctcgccgac cactaccagc
     2941 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagccatc
     3001 agtccaaact gagcaaagac cccaacgaga agcgcgatca catggtcctg aaggagaggg
     3061 tgaccgccgc cgggattaca catgacatgg acgagctgta caagggcgga ggatctccat
     3121 cacgtttgga agaggaactg aggcgccgct taactgaacc ttccggcgag acggcatgcc
     3181 ttaagagatc tggtacctgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3241 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3301 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3361 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     3421 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     3481 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3541 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     3601 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3661 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3721 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     3781 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     3841 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     3901 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3961 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4021 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     4081 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     4141 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4201 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4261 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4321 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4381 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4441 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4501 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4561 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4621 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4681 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4741 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4801 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4861 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4921 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4981 aact
//
