LOCUS       sequence-451678-        5303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate assembly (BsmBI) of donor plasmids to knock-in 
            sfGFP using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5303)
  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 5303)
  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-sfGFP
FEATURES             Location/Qualifiers
     source          1..5303
                     /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"
     promoter        640..658
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    1371..1492
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1382..1401)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1436..1455
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1613..2290)
                     /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(1675..1698)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1873..1892)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2090..2110
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2142..2160
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2287..2296
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3131..3150)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3132..3150)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3157..3174)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3157..3173)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3166..3188)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3315..3770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3402..3421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3612..3633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3796..3900
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3901..3969,3970..4761)
                     /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(4119..4138)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4932..5303,1..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 caagctcgaa attaaccctc actaaaggga
      661 acaaaagctg ggtaccgtct cgccggactt gtacagctca tccatgccca gggtgatgcc
      721 ggcggcggtc acgaactcca gcagcaccat gtgatcgcgc ttctcgttgg ggtccttgga
      781 cagcacgctc tgggtgctca ggtagtggtt atcgggcagc agcactgggc cgtcgccgat
      841 gggggtgttc tgctggtagt gatcggccag ctgcacggag ccatcctcca cattgtggcg
      901 gatcttgaag ttggccttga tgccgttctt ctgcttatcg gcggtgatgt acacgttgtg
      961 gctgttgaag ttgtactcca gcttgtggcc caggatgttg ccatcctcct tgaaatcgat
     1021 gcccttcagc tcgatgcggt tcaccagggt atcgccctcg aacttcacct cggcgcgggt
     1081 cttgtaggtg ccgtcatcct tgaagctgat ggtgcgctcc tgcacgtagc cctcgggcat
     1141 ggcgctcttg aagaaatcgt gctgcttcat gtgatcgggg tagcggctga agcactgcac
     1201 gccgtaggtc agggtggtca ccagggtggg ccagggcacg ggcagcttgc cggtggtgca
     1261 gatgaacttc agggtcagct tgccgttgga ctagtccgct ggtactgtat ggatgtacgt
     1321 cgacgtacat ccatacagta ccagcgggta catccataca gtaccagcgg taagatacat
     1381 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     1441 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     1501 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     1561 gtaaaacctc tacaaatgtg gtatggctga ttatgatcta gagtcgcggc cgctacagga
     1621 acaggtggtg gcggccctcg gcgcgctcgt actgctccac gatggtgtag tcctcgttgt
     1681 gggaggtgat gtccagcttg gagtccacgt agtagtagcc gggcagctgc acgggcttct
     1741 tggccatgta gatggacttg aactccacca ggtagtggcc gccgtccttc agcttcaggg
     1801 ccttgtggat ctcgcccttc agcacgccgt cgcgggggta caggcgctcg gtggacgcct
     1861 cccagcccat agtcttcttc tgcattacgg ggccgtcgga ggggaagttc acgccgatga
     1921 acttcacctt gtagatgaag gagccgtcct ggagggagga gtcctgggtc acggtcacca
     1981 cgccgccgtc ctcgaagttc atcacgcgct cccacttgaa gccctcgggg aaggacagct
     2041 tcttgtagtc ggggatgtcg gcggggtgct tcacgtacac cttggagccg tactggaact
     2101 ggggggacag gatgtcccag gcgaagggca gggggccgcc cttggtcacc ttcagcttgg
     2161 cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg aactcgtggc
     2221 cgttcacgga gccctccatg cgcaccttga agcgcatgaa ctccttgatg acgtcctcgg
     2281 aggaggccat ggtggcgacc ggcttcgagc cgattgttta gcttgttcag ctgcgcttgt
     2341 ttatttgctt agctttcgct tagcgacgtg ttcactttgc ttgtttgaat tgaattgtcg
     2401 ctccgtagac gaagcgcctc tatttatact ccggcggtcg agggttcgaa atcgataagc
     2461 ttggatccta attgaattag ctctaattga attagtctct aattgaatta gatccccgcg
     2521 ttgttatgat cgtactaccc gcgttgttat gatcgtacta cgctagcgta gtacgatcat
     2581 aacaacgcgg ctacctaggg cggatcttga agttggcctt gatgccgttc ttctgcttat
     2641 cggcggtgat gtacacgttg tggctgttga agttgtactc cagcttgtgg cccaggatgt
     2701 tgccatcctc cttgaaatcg atgcccttca gctcgatgcg gttcaccagg gtatcgccct
     2761 cgaacttcac ctcggcgcgg gtcttgtagg tgccgtcatc cttgaagctg atggtgcgct
     2821 cctgcacgta gccctcgggc atggcgctct tgaagaaatc gtgctgcttc atgtgatcgg
     2881 ggtagcggct gaagcactgc acgccgtagg tcagggtggt caccagggtg ggccagggca
     2941 cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgttg gtggcgtcgc
     3001 cctcgccctc gccgcgcacg ctgaacttgt ggccgttcac gtcgccatcc agctccacca
     3061 ggatgggcac cacgccggtg aacagctcct cgcccttgga caccatagag ccgagacgag
     3121 ctccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
     3181 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     3241 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     3301 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3361 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3421 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3481 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3541 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3601 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3661 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3721 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     3781 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3841 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3901 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3961 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4021 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4081 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4141 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4201 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     4261 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     4321 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4381 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4441 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4501 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4561 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4621 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4681 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4741 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4801 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4861 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4921 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4981 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5041 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5101 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5161 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5221 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5281 gagcgaacga cctacaccga act
//
