LOCUS       sequence-351751-        4092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human codon-optimized Nlacas5 with C terminal NLS and 
            HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4092)
  AUTHORS   Tan R, Krueger RK, Gramelspacher MJ, Zhou X, Xiao Y, Ke A, Hou Z, 
            Zhang Y
  TITLE     Cas11 enables genome engineering in human cells with compact 
            CRISPR-Cas3 systems.
  JOURNAL   Mol Cell. 2022 Feb 17;82(4):852-867.e5. doi: 
            10.1016/j.molcel.2021.12.032. Epub 2022 Jan 19.
  PUBMED    35051351
REFERENCE   2  (bases 1 to 4092)
  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"; doi: 
            "10.1016/j.molcel.2021.12.032"; journalName: "Mol Cell"; date: 
            "2022-02-17- 17"; volume: "82"; issue: "4"; pages: "852-867.e5"
COMMENT     Sequence Label: pSmart-EF1a-Nla-cas5
FEATURES             Location/Qualifiers
     source          1..4092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..51)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        201..1363
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          432..1354
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1311..1331
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2029..2055
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2056..2076
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2092..2109)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2098..2209
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     terminator      2367..2396
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      2418..3005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2906..2925
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3017..3044)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3065..3880)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3788..3807
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3881..3983)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(3984..4015)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
       61 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
      121 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
      181 gttttgcgct gcttcgcgga ggctccggtg cccgtcagtg ggcagagcgc acatcgccaa
      241 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtacctag agaaggtgga
      301 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
      361 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
      421 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
      481 gcccttgcgt gccttgaatt acttccacct ggctccagta cgtgattctt gatcccgagc
      541 tggagccagg ggcgggcctt gcgctttagg agccccttcg cctcgtgctt gagttgaggc
      601 ctggcctggg cgctggggcc gtcgcgtgcg aatctggtgg caccttcgcg cctgtctcgc
      661 tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
      721 ctggcaagat agtcttgtaa atgcgggcca ggatctgcac actggtattt cggtttttgg
      781 ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggaga ggcggggcct
      841 gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt
      901 gcctggcctc gcgcagccgt gtatcgcccc gcactgggcg gcaaggctgg cccggtcggc
      961 accagttgcg tgagcggaaa gatggccgct tcccggccct gctccagggg gctcaaaatg
     1021 gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaggggcctt
     1081 tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct
     1141 cgattagttc tggagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc
     1201 gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat
     1261 gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
     1321 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaacaccgc caccatgcgg
     1381 ttcatcctgg aaatcagcgg cgacctggcc tgcttcacaa gaagcgagct gaaggtcgag
     1441 cgggtgtcat accctgtgat cacccctagc gccgccagaa acatcctgat ggccattctg
     1501 tggaagcccg ccatcagatg gaaggtgctg aagatcgaga tcctgaagcc tatccagtgg
     1561 accaacatcc ggcggaacga agtgggcacc aagatgagcg agagaagcgg cagcctgtac
     1621 atcgaggaca acagacagca gcgggcctcc atgctgctga aggatgtggc ctatagaatc
     1681 cacgccgact tcgacatgac aagcgaggcc ggcgagagcg acaactacgt gaagttcgcc
     1741 gagatgttca agcggagagc caagaagggc cagtacttcc accagcctta cctgggctgc
     1801 agagagttcc cctgcgactt cagactgctg gaaaaggccg aggatggcct gcctctggaa
     1861 gatatcaccc aggacttcgg cttcatgctg tacgacatgg acttcagcaa gagcgacccc
     1921 agagacagca acaacgccga gcctatgttc taccagtgca aggccgtgaa cggcgtgatc
     1981 actgtgcctc cagccgatag cgaggaagtg aagagaggca gcgtcggcta cccctacgat
     2041 gtgcctgatt acgcccctaa gaaaaagcgg aaagtgtgac tcgctgatca gcctcgactg
     2101 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2161 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2221 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2281 aagacaatag caggaacgaa ttcaagcttg atatcattca ggacgagcct cagactccag
     2341 cgtaactgga ctgcaatcaa ctcactggct caccttcacg ggtgggcctt tcttcggtag
     2401 aaaatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2461 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2521 ccgaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     2581 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2641 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2701 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2761 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2821 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2881 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2941 ttcgccacct ctgacttgag catcgatttt tgtgatgctc gtcagggggg cggagcctat
     3001 ggaaaaacgc cagcaacgca gaaaggccca cccgaaggtg agccaggtga ttacatttgg
     3061 gccctcagaa aaactcatcg agcatcaagt gaaactgcaa tttattcata tcaggattat
     3121 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     3181 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     3241 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     3301 cgactgaatc cggtgagaat ggcaaaagct tatgcatttc tttccagact tgttcaacag
     3361 gccagccatt acgctcgtca tcaaaatcac tcgcaccaac caaaccgtta ttcattcgtg
     3421 attgcgcctg agcgagacga aatacgcgat cgccgttaaa aggacaatta caaacaggaa
     3481 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     3541 gatattcttc taatacctgg aatgctgttt tccctgggat cgcagtggtg agtaaccatg
     3601 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     3661 agtttagcct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     3721 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     3781 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     3841 gcggcctcga gcaagacgtt tcccgttgaa tatggctcat tttagcttcc ttagctcctg
     3901 aaaatctcga taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt
     3961 tggaacctct tacgtgccga tcaagtcaaa agcctccggt cggaggcttt tgactttctg
     4021 ctatggaggt caggtatgat ttaaatggtc agtattgagc gatatctaga gaattcgtcc
     4081 gacggatcgg ga
//
