LOCUS       sequence-341879-        5447 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 NanoLuc 
            and selection with Puromycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5447)
  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 5447)
  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-NanoLuc-Puromycin
FEATURES             Location/Qualifiers
     source          1..5447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             954..1259
                     /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(1354..1370)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1354..1370)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1367..1389)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1378..1394
                     /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(1402..1408,1409..1426,1427..1432))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1447..1468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1585..1602)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1756..2344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1836..1855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2515..3306,3307..3375))
                     /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     3138..3157
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3376..3480)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3548..3566
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3604..3626)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3726..3745
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3939..3961
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3953..3970
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3954..3970
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4307..4612
                     /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             5190..5207
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5223..5279
                     /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 acccgggtgg ggctggacat aggcaaagtc tgggtcgccg acgatggcgc cgcggtggcc
       61 gtgtggacca cgccggagag cgtcgaggcc ggggctgtgt ttgcagagat cgggccccgg
      121 atggcggagc tgtctggcag ccgactggct gcccagcagc agatggaggg actcctggca
      181 ccccacaggc cgaaggagcc cgcttggttt ttggccacag ttggggttag cccggaccac
      241 cagggcaagg gactcggttc cgcagtagtc ttgccagggg tggaggcagc cgagagggca
      301 ggagtcccgg ctttcctcga gactagtgcc ccacgaaatc tgccctttta cgagcgcctt
      361 ggatttaccg tcaccgccga tgtcgaagtt ccagaaggac cgaggacttg gtgcatgaca
      421 agaaaaccgg gggcatctag ataaattcgt cagtagggtt gtaaaggttt ttcttttcct
      481 gagaaaacaa ccttttgttt tctcaggttt tgctttttgg cctttcccta gcttttaaaa
      541 aaaaaaaagc aaaagacgct ggtggctggc actcctggtt tccaggacgg ggttcaagtc
      601 cctgcggtgt ctttgcttgg atccggctta ctaaaagcca gataacagta tgcgtatttg
      661 cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag tatgtcaaaa
      721 agaggtatgc tatgaagcag cgtattacag tgacagttga cagcgacagc tatcagttgc
      781 tcaaggcata tatgatgtca atatctccgg tctggtaagc acaaccatgc agaatgaagc
      841 ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc
      901 ccggtttatt gaaatgaacg gctcttttgc tgacgagaac aggggctggt gaaatgcagt
      961 ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg
     1021 atattattga cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt
     1081 cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca
     1141 tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc
     1201 tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa
     1261 tgtcaggctc ccttatacac agccagtctg caggtcgacg gatcccgggc ccgtcgactg
     1321 cagaggcctg catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     1381 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     1441 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     1501 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1561 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     1621 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     1681 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1741 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1801 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1861 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1921 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     1981 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2041 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2101 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2161 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2221 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2281 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2341 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2401 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2461 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     2521 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     2581 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     2641 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     2701 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     2761 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     2821 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     2881 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     2941 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3001 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3061 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3121 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     3181 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     3241 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     3301 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     3361 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     3421 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     3481 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     3541 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     3601 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     3661 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     3721 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     3781 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     3841 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     3901 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     3961 gacggccagt gggtaccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg
     4021 atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta
     4081 tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc
     4141 atatatgatg tcaatatctc cggtctggta agcacaacca tgcagaatga agcccgtcgt
     4201 ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt
     4261 attgaaatga acggctcttt tgctgacgag aacaggggct ggtgaaatgc agtttaaggt
     4321 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat
     4381 tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa
     4441 agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac
     4501 caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca
     4561 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg
     4621 ctcccttata cacagccagt ctgcaggtcg acggtaccca aggcggtgga gaattcatgg
     4681 tgtttacgtt ggaggacttc gtgggagatt ggagacaaac cgccggctac aatctggatc
     4741 aggttctgga gcaaggtggg gtgagctccc tttttcagaa ccttggagtt tctgtcacgc
     4801 ccatccaaag gattgttctg tccggggaga acggcctgaa gatagatatc cacgtgatca
     4861 tcccttatga gggccttagc ggcgatcaaa tgggccagat cgagaaaatt ttcaaggttg
     4921 tgtatcctgt cgacgaccat catttcaaag tcatccttca ctatggaaca ctggtcatcg
     4981 acggggtgac accaaacatg atagattact tcggaaggcc ctatgagggt attgcggtat
     5041 tcgatggcaa gaaaattact gtaacaggta cactgtggaa cggaaataag atcatcgacg
     5101 agcggctgat caatcccgat ggtagtctgc ttttcagagt aaccatcaat ggagttaccg
     5161 ggtggcgact ctgcgagagg attttggctc accatcacca tcaccatgaa ttcggatctg
     5221 gagcaacaaa cttctcacta ctcaaacaag caggtgacgt ggaggagaat cccgggcctt
     5281 ctagaatgac agagtataaa cccactgtga gattggcaac cagggatgac gtgcccagag
     5341 ccgtgcggac cctcgcagcc gcgtttgccg actatcctgc aaccaggcat acggtcgacc
     5401 cagatcggca tatcgagaga gtgactgaat tgcaggagct tttcctg
//
