LOCUS       sequence-410641-        4979 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 4979)
  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 4979)
  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-N
FEATURES             Location/Qualifiers
     source          1..4979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      104..558
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(191..210)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     400..421
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        584..688
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(689..757,758..1549)
                     /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(907..926)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1720..2308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2209..2228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2462..2479
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2613..2837
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     protein_bind    complement(2900..2933)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2936..3643
                     /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      3191..3200
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3644..3700
                     /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(3701..3734)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4331..4387
                     /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"
     CDS             join(4397..4420,4421..4456,4457..4480)
                     /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             4493..4531
                     /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             4547..4594
                     /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             4604..4669
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4670..4687
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     mobile_element  4734..4899
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 ttttctctgt cacagaatga aaatttttct gtcatctctt cgttattaat gtttgtaatt
       61 gactgaatat caacgcttat ttgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
      121 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
      181 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
      241 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
      301 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
      361 gtttttcgcc ctttgacgtt ggagtccacg ttcttaatag tggactcttg ttccaaactg
      421 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
      481 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      541 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      601 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      661 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      721 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      781 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      841 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      901 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
      961 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1021 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1081 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1141 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1201 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1261 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1321 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1381 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1441 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1501 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1561 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1621 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1681 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1741 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1801 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1861 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1921 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1981 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2041 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2101 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2161 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2221 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2281 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2341 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2401 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2461 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2521 tctgtgcggt atttcacacc gcatagacca gccgcgtaac ctggcaaaat cggttacggt
     2581 tgagtaataa atggatgccc tgcgtaagcg ggtgtgggcg gacaataaag tcttaaactg
     2641 aacaaaatag atctaaacta tgacaataaa gtcttaaact agacagaata gttgtaaact
     2701 gaaatcagtc cagttatgct gtgaaaaagc atactggact tttgttatgg ctaaagcaaa
     2761 ctcttcattt tctgaagtgc aaattgcccg tcgtattaaa gaggggcgtg gccaagggca
     2821 tggtaaagac tatattcgcg gcgttgtgac aatttaccga acaactcgtt taaacgcggc
     2881 cgctttcgaa tctagaatga taacttcgta tagcatacat tatacgaagt tatctatggt
     2941 gagcaagggc gaggaggata acatggcctc tctcccagcg acacatgagt tacacatctt
     3001 tggctccatc aacggtgtgg actttgacat ggtgggtcag ggcaccggca atccaaatga
     3061 tggttatgag gagttaaacc tgaagtccac caagggtgac ctccagttct ccccctggat
     3121 tctggtccct catatcgggt atggcttcca tcagtacctg ccctaccctg acgggatgtc
     3181 gcctttccag gccgccatgg tagatggctc cggataccaa gtccatcgca caatgcagtt
     3241 tgaagatggt gcctccctta ctgttaacta ccgctacacc tacgagggaa gccacatcaa
     3301 aggagaggcc caggtgaagg ggactggttt ccctgctgac ggtcctgtga tgaccaactc
     3361 gctgaccgct gcggactggt gcaggtcgaa gaagacttac cccaacgaca aaaccatcat
     3421 cagtaccttt aagtggagtt acaccactgg aaatggcaag cgctaccgga gcactgcgcg
     3481 gaccacctac acctttgcca agccaatggc ggctaactat ctgaagaacc agccgatgta
     3541 cgtgttccgt aagacggagc tcaagcactc caagaccgag ctcaacttca aggagtggca
     3601 aaaggccttt accgatgtga tgggcatgga cgagctgtac aaggctacta acttcagcct
     3661 gctgaagcaa gctggagacg tggaggagaa ccctggacct ataacttcgt atagcataca
     3721 ttatacgaag ttatctatga cagagtataa accaacggtt cggctcgcaa ctcgcgacga
     3781 tgtgccccga gcagttagga ctttggccgc ggcgttcgca gactatccag cgacgaggca
     3841 caccgtagat ccggataggc atattgaacg ggtcaccgag cttcaggaac tttttctcac
     3901 gagagttggc cttgatatcg gaaaggtatg ggtagcggac gacggggctg ctgtagcggt
     3961 ctggaccacg ccagaatcag tggaagcggg ggcggtattc gcagaaattg gtccgaggat
     4021 ggcggagttg tccgggtctc gactggctgc ccagcaacag atggagggtc ttctcgctcc
     4081 gcaccgacca aaggaaccgg cttggttcct ggctacagtt ggcgtttcac cagatcacca
     4141 aggtaaagga cttggaagcg cagtcgtcct tccgggggtt gaggcagcgg aacgggcagg
     4201 tgtccccgcg ttcttggaga ccagtgctcc taggaacctc cctttctacg agcgacttgg
     4261 gttcacggtc acagctgatg tagaggttcc agagggcccc aggacttggt gtatgaccag
     4321 gaaaccgggt gccacgaatt tcagcctgct taagcaagcc ggtgacgttg aagagaatcc
     4381 aggccccccg ggttcctggt cccaccccca atttgaaaag ggtggaggaa gtggaggtgg
     4441 ttccggaggt agcgcgtgga gtcacccaca attcgagaaa gggagtggac cgagccgcct
     4501 ggaagaagaa ctgcgccgcc gcctgaccga accgggttca ggatcccgag atcatatggt
     4561 gctccacgaa tacgtgaatg cggcaggcat cacaggttcc ccggattaca aagaccatga
     4621 cggggattat aaagaccacg atattgatta taaagacgac gatgacaaat tggtgccgcg
     4681 gggcagcggt tcagaattct aagcttctcg agcaattggt ttaaacagat ccgaaccaga
     4741 taagtgaaat ctagttccaa actattttgt catttttaat tttcgtatta gcttacgacg
     4801 ctacacccag ttcccatcta ttttgtcact cttccctaaa taatccttaa aaactccatt
     4861 tccacccctc ccagttccca actattttgt ccgcccacag cggggcattt ttcttcctgt
     4921 tatgttttta atcaaacatc ctgccaactc catgtgacaa accgtcatct tcggctact
//
