LOCUS       sequence-341910-        5717 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid to clone recombination arms to create homologous 
            recombination donor vector for C-terminal gene tagging with mClover3
            and selection with Puromycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5717)
  AUTHORS   Perez-Leal O, Nixon-Abell J, Barrero CA, Gordon JC, Oesterling J, 
            Rico MC
  TITLE     Multiplex Gene Tagging with CRISPR-Cas9 for Live-Cell Microscopy and
            Application to Study the Role of SARS-CoV-2 Proteins in Autophagy, 
            Mitochondrial Dynamics, and Cell Growth.
  JOURNAL   CRISPR J. 2021 Nov 30. doi: 10.1089/crispr.2021.0041.
  PUBMED    34847745
REFERENCE   2  (bases 1 to 5717)
  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: "CRISPR J. 
            2021 Nov 30. doi: 10.1089/crispr.2021.0041."
COMMENT     Sequence Label: FASTHDR-Cterm-mClover3-Puromycin
FEATURES             Location/Qualifiers
     source          1..5717
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             732..1037
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             1819..1884
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1885..1902
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1918..1974
                     /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             3096..3401
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3496..3512)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3496..3512)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3509..3531)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3520..3536
                     /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(3544..3550,3551..3568,3569..3574))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3589..3610
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3727..3744)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3898..4486)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3978..3997)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4657..5448,5449..5517))
                     /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     5280..5299
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5518..5622)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5690..5708
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgggta ccggcttact aaaagccaga
      421 taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac tgatatgtat
      481 acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg acagttgaca
      541 gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac
      601 aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga
      661 agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag
      721 gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt
      781 ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg
      841 ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg
      901 gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg
      961 gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga
     1021 tgttctgggg aatataaatg tcaggctccc ttatacacag ccagtctgca ggtcgacggt
     1081 acccaaggcg gtggagaatt catggttagc aaaggagagg agctgtttac gggcgttgtt
     1141 ccgattctgg tggagctgga cggagacgtc aacggtcata agtttagtgt tcgaggtgaa
     1201 ggtgaaggtg atgcgacgaa tggcaaactg actctgaaat tcatttgtac gacagggaaa
     1261 cttcctgtac cctggccgac acttgtgaca acattcggat atggggtcgc atgtttcagc
     1321 aggtatccgg atcatatgaa gcaacatgac tttttcaaga gcgccatgcc agaagggtat
     1381 gttcaggaga ggacgatttc atttaaggac gacggaacct ataagacgcg ggcggaagtg
     1441 aaatttgagg gtgacaccct tgtcaataga attgaactga aggggatcga ttttaaggag
     1501 gacggaaaca ttctcggaca taaattggag tacaatttca actcccatta tgtgtatata
     1561 actgctgata agcaaaagaa ctgcataaag gccaatttta agataaggca caatgttgaa
     1621 gacggatcag tacagttggc ggatcattat cagcaaaata cacctatagg ggacggtccc
     1681 gtgcttttgc ctgataacca ctatttgagt catcagtcta aattgtccaa agatcccaac
     1741 gagaaaaggg accacatggt actccttgag tttgtaacgg ctgcgggcat tacccatggt
     1801 atggacgagc tttacaaaga ctataaggac cacgacggag actacaagga tcatgatatt
     1861 gattacaaag acgatgacga taagcaccat caccatcacc atgaattcgg atctggagca
     1921 acaaacttct cactactcaa acaagcaggt gacgtggagg agaatcccgg gccttctaga
     1981 atgacagagt ataaacccac tgtgagattg gcaaccaggg atgacgtgcc cagagccgtg
     2041 cggaccctcg cagccgcgtt tgccgactat cctgcaacca ggcatacggt cgacccagat
     2101 cggcatatcg agagagtgac tgaattgcag gagcttttcc tgacccgggt ggggctggac
     2161 ataggcaaag tctgggtcgc cgacgatggc gccgcggtgg ccgtgtggac cacgccggag
     2221 agcgtcgagg ccggggctgt gtttgcagag atcgggcccc ggatggcgga gctgtctggc
     2281 agccgactgg ctgcccagca gcagatggag ggactcctgg caccccacag gccgaaggag
     2341 cccgcttggt ttttggccac agttggggtt agcccggacc accagggcaa gggactcggt
     2401 tccgcagtag tcttgccagg ggtggaggca gccgagaggg caggagtccc ggctttcctc
     2461 gagactagtg ccccacgaaa tctgcccttt tacgagcgcc ttggatttac cgtcaccgcc
     2521 gatgtcgaag ttccagaagg accgaggact tggtgcatga caagaaaacc gggggcatct
     2581 agataaattc gtcagtaggg ttgtaaaggt ttttcttttc ctgagaaaac aaccttttgt
     2641 tttctcaggt tttgcttttt ggcctttccc tagcttttaa aaaaaaaaaa gcaaaagacg
     2701 ctggtggctg gcactcctgg tttccaggac ggggttcaag tccctgcggt gtctttgctt
     2761 ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     2821 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc
     2881 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     2941 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     3001 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     3061 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata
     3121 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg
     3181 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     3241 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     3301 ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     3361 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
     3421 acagccagtc tgcaggtcga cggatcccgg gcccgtcgac tgcagaggcc tgcatgcaag
     3481 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3541 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3601 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3661 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3721 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3781 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3841 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3901 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3961 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4021 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4081 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4141 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4201 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4261 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4321 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4381 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4441 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4501 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4561 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4621 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4681 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4741 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4801 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4861 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4921 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4981 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5041 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5101 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5161 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5221 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5281 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5341 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5401 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5461 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5521 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5581 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5641 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     5701 cacgaggccc tttcgtc
//
