LOCUS       sequence-452264-        3975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate assembly of new SEED cassettes via Golden Gate 
            assembly (BsaI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3975)
  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 3975)
  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
FEATURES             Location/Qualifiers
     source          1..3975
                     /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"
     polyA_signal    755..876
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(766..785)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     820..839
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(997..1674)
                     /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(1059..1082)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1257..1276)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1474..1494
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1526..1544
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1671..1680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2023..2040)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2023..2039)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2032..2054)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2248..2267)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2367..2389
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2427..2445)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2513..2617
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2618..3433
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRFAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2691..2710)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(3604..3975,1..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 agcggaacag 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 atgctcgagc caagctcggc gcgcccgtct cgccggaacc
      661 gagaccggtc tctactagtc cgctggtact gtatggatgt acgtcgacgt acatccatac
      721 agtaccagcg ggtacatcca tacagtacca gcggtaagat acattgatga gtttggacaa
      781 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
      841 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
      901 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa
      961 tgtggtatgg ctgattatga tctagagtcg cggccgctac aggaacaggt ggtggcggcc
     1021 ctcggcgcgc tcgtactgct ccacgatggt gtagtcctcg ttgtgggagg tgatgtccag
     1081 cttggagtcc acgtagtagt agccgggcag ctgcacgggc ttcttggcca tgtagatgga
     1141 cttgaactcc accaggtagt ggccgccgtc cttcagcttc agggccttgt ggatctcgcc
     1201 cttcagcacg ccgtcgcggg ggtacaggcg ctcggtggac gcctcccagc ccatagtctt
     1261 cttctgcatt acggggccgt cggaggggaa gttcacgccg atgaacttca ccttgtagat
     1321 gaaggagccg tcctggaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa
     1381 gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcttgt agtcggggat
     1441 gtcggcgggg tgcttcacgt acaccttgga gccgtactgg aactgggggg acaggatgtc
     1501 ccaggcgaag ggcagggggc cgcccttggt caccttcagc ttggcggtct gggtgccctc
     1561 gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc
     1621 catgcgcacc ttgaagcgca tgaactcctt gatgacgtcc tcggaggagg ccatggtggc
     1681 gaccggcttc gagccgattg tttagcttgt tcagctgcgc ttgtttattt gcttagcttt
     1741 cgcttagcga cgtgttcact ttgcttgttt gaattgaatt gtcgctccgt agacgaagcg
     1801 cctctattta tactccggcg gtcgagggtt cgaaatcgat aagcttggat cctaattgaa
     1861 ttagctctaa ttgaattagt ctctaattga attagatccc cgcgttgtta tgatcgtact
     1921 acccgcgttg ttatgatcgt actacgctag cgtagtacga tcataacaac gcggctacct
     1981 agggtagaga ccggtctcag agccgagacg acgcgtcaat tcactggccg tcgttttaca
     2041 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2101 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2161 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2221 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2281 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2341 cgcttacaga caagctgtga cagtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2401 atcaccgaaa cgcgcgagac aaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2461 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2521 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2581 ctgataaatg cttcaataat attgaaaaag gaagagtatg agccatattc aacgggaaac
     2641 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     2701 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     2761 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     2821 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     2881 ccgtactcct gatgatgcat ggttactcac cactgcgatc cctgggaaaa cagcattcca
     2941 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     3001 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     3061 tttcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     3121 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     3181 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     3241 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     3301 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     3361 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     3421 tgagtttttc taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3481 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3541 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3601 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3661 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3721 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     3781 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3841 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3901 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3961 gacctacacc gaact
//
