LOCUS       sequence-452263-        5360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Permits GoldenGate assembly (BsmBI) of donor plasmids to knock-in 
            sfGFP:ALFA using the SEED/Harvest method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5360)
  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 5360)
  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:ALFA
FEATURES             Location/Qualifiers
     source          1..5360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1480..1489
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1486..2163
                     /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(1616..1634)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1666..1686)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1884..1903
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2078..2101
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2284..2405
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2321..2340)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2375..2394
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             3104..3142
                     /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"
     promoter        complement(3175..3193)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3214..3230)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3214..3230)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3227..3249)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3238..3254
                     /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)."
     promoter        complement(join(3262..3268,3269..3286,3287..3292))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3307..3328
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3445..3462)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3616..4204)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3696..3715)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4375..5166,5167..5235))
                     /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     4998..5017
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5236..5340)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgt
      661 ctcggctcta tggtgtccaa gggcgaggag ctgttcaccg gcgtggtgcc catcctggtg
      721 gagctggatg gcgacgtgaa cggccacaag ttcagcgtgc gcggcgaggg cgagggcgac
      781 gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      841 tggcccaccc tggtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgat
      901 cacatgaagc agcacgattt cttcaagagc gccatgcccg agggctacgt gcaggagcgc
      961 accatcagct tcaaggatga cggcacctac aagacccgcg ccgaggtgaa gttcgagggc
     1021 gataccctgg tgaaccgcat cgagctgaag ggcatcgatt tcaaggagga tggcaacatc
     1081 ctgggccaca agctggagta caacttcaac agccacaacg tgtacatcac cgccgataag
     1141 cagaagaacg gcatcaaggc caacttcaag atccgcccta ggtagccgcg ttgttatgat
     1201 cgtactacgc tagcgtagta cgatcataac aacgcgggta gtacgatcat aacaacgcgg
     1261 ggatctaatt caattagaga ctaattcaat tagagctaat tcaattagga tccaagctta
     1321 tcgatttcga accctcgacc gccggagtat aaatagaggc gcttcgtcta cggagcgaca
     1381 attcaattca aacaagcaaa gtgaacacgt cgctaagcga aagctaagca aataaacaag
     1441 cgcagctgaa caagctaaac aatcggctcg aagccggtcg ccaccatggc ctcctccgag
     1501 gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg agggctccgt gaacggccac
     1561 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     1621 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc
     1681 cagtacggct ccaaggtgta cgtgaagcac cccgccgaca tccccgacta caagaagctg
     1741 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     1801 accgtgaccc aggactcctc cctccaggac ggctccttca tctacaaggt gaagttcatc
     1861 ggcgtgaact tcccctccga cggccccgta atgcagaaga agactatggg ctgggaggcg
     1921 tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca caaggccctg
     1981 aagctgaagg acggcggcca ctacctggtg gagttcaagt ccatctacat ggccaagaag
     2041 cccgtgcagc tgcccggcta ctactacgtg gactccaagc tggacatcac ctcccacaac
     2101 gaggactaca ccatcgtgga gcagtacgag cgcgccgagg gccgccacca cctgttcctg
     2161 tagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     2221 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     2281 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2341 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2401 tcttaccgct ggtactgtat ggatgtaccc gctggtactg tatggatgta cgtcgacgta
     2461 catccataca gtaccagcgg actagtccaa cggcaagctg accctgaagt tcatctgcac
     2521 caccggcaag ctgcccgtgc cctggcccac cctggtgacc accctgacct acggcgtgca
     2581 gtgcttcagc cgctaccccg atcacatgaa gcagcacgat ttcttcaaga gcgccatgcc
     2641 cgagggctac gtgcaggagc gcaccatcag cttcaaggat gacggcacct acaagacccg
     2701 cgccgaggtg aagttcgagg gcgataccct ggtgaaccgc atcgagctga agggcatcga
     2761 tttcaaggag gatggcaaca tcctgggcca caagctggag tacaacttca acagccacaa
     2821 cgtgtacatc accgccgata agcagaagaa cggcatcaag gccaacttca agatccgcca
     2881 caatgtggag gatggctccg tgcagctggc cgatcactac cagcagaaca cccccatcgg
     2941 cgacggccca gtgctgctgc ccgataacca ctacctgagc acccagagcg tgctgtccaa
     3001 ggaccccaac gagaagcgcg atcacatggt gctgctggag ttcgtgaccg ccgccggcat
     3061 caccctgggc atggatgagc tgtacaaggg cggaggatct ccatcacgtt tggaagagga
     3121 actgaggcgc cgcttaactg aaccttccgg cgagacggta cccagctttt gttcccttta
     3181 gtgagggtta atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3241 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     3301 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3361 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3421 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3481 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3541 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3601 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3661 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3721 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3781 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3841 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3901 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3961 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4021 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4081 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4141 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4201 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4261 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4321 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4381 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4441 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4501 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4561 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4621 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4681 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4741 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4801 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4861 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4921 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4981 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5041 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5101 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5161 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5221 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5281 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5341 cacatttccc cgaaaagtgc
//
