LOCUS       sequence-414780-        4976 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR donor template with Puromycin resistance and mNeonGreen marker 
            flanked by two multiple cloning sites (MCS) for insertion of left 
            and right homology arms (L- and R-arms) for the targeted locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4976)
  AUTHORS   Choi W, Wu H, Yserentant K, Huang B, Cheng Y
  TITLE     Efficient tagging of endogenous proteins in human cell lines for 
            structural studies by single-particle cryo-EM.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Aug;120(31):e2302471120. doi: 
            10.1073/pnas.2302471120. Epub 2023 Jul 24.
  PUBMED    37487103
REFERENCE   2  (bases 1 to 4976)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2023-08"; volume: "120(31):e2302471120. 
            doi"; pages: " 10.1073/pnas.2302471120. Epub 2023 Jul 24"
COMMENT     Sequence Label: pYC-C
FEATURES             Location/Qualifiers
     source          1..4976
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             36..743
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      291..300
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     mobile_element  823..988
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      1172..1626
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1259..1278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1468..1489
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1652..1756
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1757..1825,1826..2617)
                     /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(1975..1994)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2788..3376
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3277..3296
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3530..3547
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  3681..3905
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             3968..3985
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3992..4057
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4070..4117
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             4127..4165
                     /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"
     CDS             join(4175..4198,4199..4234,4235..4258)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             4268..4324
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    complement(4919..4952)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             join(4955..4976,1..35)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
ORIGIN
        1 agcaagctgg agacgtggag gagaaccctg gacctatggt gagcaagggc gaggaggata
       61 acatggcctc tctcccagcg acacatgagt tacacatctt tggctccatc aacggtgtgg
      121 actttgacat ggtgggtcag ggcaccggca atccaaatga tggttatgag gagttaaacc
      181 tgaagtccac caagggtgac ctccagttct ccccctggat tctggtccct catatcgggt
      241 atggcttcca tcagtacctg ccctaccctg acgggatgtc gcctttccag gccgccatgg
      301 tagatggctc cggataccaa gtccatcgca caatgcagtt tgaagatggt gcctccctta
      361 ctgttaacta ccgctacacc tacgagggaa gccacatcaa aggagaggcc caggtgaagg
      421 ggactggttt ccctgctgac ggtcctgtga tgaccaactc gctgaccgct gcggactggt
      481 gcaggtcgaa gaagacttac cccaacgaca aaaccatcat cagtaccttt aagtggagtt
      541 acaccactgg aaatggcaag cgctaccgga gcactgcgcg gaccacctac acctttgcca
      601 agccaatggc ggctaactat ctgaagaacc agccgatgta cgtgttccgt aagacggagc
      661 tcaagcactc caagaccgag ctcaacttca aggagtggca aaaggccttt accgatgtga
      721 tgggcatgga cgagctgtac aagataactt cgtatagcat acattatacg aagttatctt
      781 gagaattcta agcttctcga gcaattggtt taaacagatc cgaaccagat aagtgaaatc
      841 tagttccaaa ctattttgtc atttttaatt ttcgtattag cttacgacgc tacacccagt
      901 tcccatctat tttgtcactc ttccctaaat aatccttaaa aactccattt ccacccctcc
      961 cagttcccaa ctattttgtc cgcccacagc ggggcatttt tcttcctgtt atgtttttaa
     1021 tcaaacatcc tgccaactcc atgtgacaaa ccgtcatctt cggctacttt ttctctgtca
     1081 cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga ctgaatatca
     1141 acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     1201 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1261 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1321 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1381 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1441 ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga acaacactca
     1501 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1561 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1621 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1681 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1741 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1801 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1861 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1921 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1981 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     2041 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     2101 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     2161 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2221 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2281 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     2341 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2401 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2461 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2521 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2581 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2641 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2701 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2761 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2821 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2881 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     2941 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3001 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3061 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3121 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3181 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3241 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3301 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3361 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3421 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3481 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3541 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3601 ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg agtaataaat
     3661 ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa caaaatagat
     3721 ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga aatcagtcca
     3781 gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact cttcattttc
     3841 tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg gtaaagacta
     3901 tattcgcggc gttgtgacaa tttaccgaac aactcgttta aacgcggccg ctttcgaatc
     3961 tagaatgttg gtgccgcggg gcagcggttc agattacaaa gaccatgacg gggattataa
     4021 agaccacgat attgattata aagacgacga tgacaaaggt tcaggatccc gagatcatat
     4081 ggtgctccac gaatacgtga atgcggcagg catcacaggt tccccgagcc gcctggaaga
     4141 agaactgcgc cgccgcctga ccgaaccggg ttcctggtcc cacccccaat ttgaaaaggg
     4201 tggaggaagt ggaggtggtt ccggaggtag cgcgtggagt cacccacaat tcgagaaagg
     4261 gagtggagcc acgaatttca gcctgcttaa gcaagccggt gacgttgaag agaatccagg
     4321 ccccatgaca gagtataaac caacggttcg gctcgcaact cgcgacgatg tgccccgagc
     4381 agttaggact ttggccgcgg cgttcgcaga ctatccagcg acgaggcaca ccgtagatcc
     4441 ggataggcat attgaacggg tcaccgagct tcaggaactt tttctcacga gagttggcct
     4501 tgatatcgga aaggtatggg tagcggacga cggggctgct gtagcggtct ggaccacgcc
     4561 agaatcagtg gaagcggggg cggtattcgc agaaattggt ccgaggatgg cggagttgtc
     4621 cgggtctcga ctggctgccc agcaacagat ggagggtctt ctcgctccgc accgaccaaa
     4681 ggaaccggct tggttcctgg ctacagttgg cgtttcacca gatcaccaag gtaaaggact
     4741 tggaagcgca gtcgtccttc cgggggttga ggcagcggaa cgggcaggtg tccccgcgtt
     4801 cttggagacc agtgctccta ggaacctccc tttctacgag cgacttgggt tcacggtcac
     4861 agctgatgta gaggttccag agggccccag gacttggtgt atgaccagga aaccgggtat
     4921 aacttcgtat agcatacatt atacgaagtt atctgctact aacttcagcc tgctga
//
