LOCUS       sequence-341878-        5423 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 mRuby3 
            and selection with Zeocin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5423)
  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 5423)
  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-mRuby3-Zeo
FEATURES             Location/Qualifiers
     source          1..5423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1974..1992
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2030..2052)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2152..2171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2365..2387
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2379..2396
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2380..2396
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2733..3038
                     /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             3103..3813
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     CDS             3814..3831
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3847..3903
                     /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             4803..5108
                     /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(5203..5219)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5203..5219)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5216..5238)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5227..5243
                     /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(5251..5257,5258..5275,5276..5281))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5296..5317
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     1981 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     2041 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     2101 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     2161 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     2221 gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     2281 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     2341 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgggt
     2401 accggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata
     2461 agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc
     2521 gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa
     2581 tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg
     2641 ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaacgg
     2701 ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac acctataaaa
     2761 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc
     2821 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     2881 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
     2941 gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     3001 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca
     3061 gccagtctgc aggtcgacgg tacccaaggc ggtggagaat tcatggtgtc taagggcgaa
     3121 gagctgatca aggaaaatat gcgtatgaag gtggtcatgg aaggttcggt caacggccac
     3181 caattcaaat gcacaggtga aggagaaggc agaccgtacg agggagtgca aaccatgagg
     3241 atcaaagtca tcgagggagg acccctgcca tttgcctttg acattcttgc cacgtcgttc
     3301 atgtatggca gccgtacctt tatcaagtac ccggccgaca tccctgattt ctttaaacag
     3361 tcctttcctg agggttttac ttgggaaaga gttacgagat acgaagatgg tggagtcgtc
     3421 accgtcacgc aggacaccag ccttgaggat ggcgagctcg tctacaacgt caaggtcaga
     3481 ggggtaaact ttccctccaa tggtcccgtg atgcagaaga agaccaaggg ttgggagcct
     3541 aatacagaga tgatgtatcc agcagatggt ggtctgagag gatacactga catcgcactg
     3601 aaagttgatg gtggtggcca tctgcactgc aacttcgtga caacttacag gtcaaaaaag
     3661 accgtcggga acatcaagat gcccggtgtc catgccgttg atcaccgcct ggaaaggatc
     3721 gaggagagtg acaatgaaac ctacgtagtg caaagagaag tggcagttgc caaatacagc
     3781 aaccttggtg gtggcatgga cgagctgtac aagcaccatc accatcacca tgaattcgga
     3841 tctggagcaa caaacttctc actactcaaa caagcaggtg acgtggagga gaatcccggg
     3901 ccttctagaa tggcgaaact gacaagcgcc gtgcctgttt tgacagcaag agacgtggca
     3961 ggtgcagttg agttttggac tgaccggctt gggttttcac gggacttcgt ggaagatgac
     4021 ttcgcaggtg tcgtgaggga tgatgtaacg ctgttcataa gcgccgtcca ggatcaagtc
     4081 gtcccggata acactttggc atgggtgtgg gttagggggc ttgacgagct ttacgcggag
     4141 tggagcgagg ttgtatctac gaatttccga gacgctagtg gcccggctat gactgaaatt
     4201 ggtgaacagc catggggtcg ggagtttgcg ttgcgcgatc ctgcaggaaa ttgcgtccac
     4261 tttgtagcgg aggaacagga ctaatctaga taaattcgtc agtagggttg taaaggtttt
     4321 tcttttcctg agaaaacaac cttttgtttt ctcaggtttt gctttttggc ctttccctag
     4381 cttttaaaaa aaaaaaagca aaagacgctg gtggctggca ctcctggttt ccaggacggg
     4441 gttcaagtcc ctgcggtgtc tttgcttgga tccggcttac taaaagccag ataacagtat
     4501 gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta tacccgaagt
     4561 atgtcaaaaa gaggtatgct atgaagcagc gtattacagt gacagttgac agcgacagct
     4621 atcagttgct caaggcatat atgatgtcaa tatctccggt ctggtaagca caaccatgca
     4681 gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc
     4741 tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct gacgagaaca ggggctggtg
     4801 aaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg
     4861 tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg gccagtgcac
     4921 gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa
     4981 gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag
     5041 tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg
     5101 gaatataaat gtcaggctcc cttatacaca gccagtctgc aggtcgacgg atcccgggcc
     5161 cgtcgactgc agaggcctgc atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt
     5221 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5281 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5341 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5401 aggcggtttg cgtattgggc gct
//
