LOCUS       sequence-351739-        4299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human codon-optimized Nlacas7 with C terminal NLS and 
            HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4299)
  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 4299)
  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-cas7
FEATURES             Location/Qualifiers
     source          1..4299
                     /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             2236..2262
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2263..2283
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2299..2316)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2305..2416
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     terminator      2574..2603
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      2625..3212
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3113..3132
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3224..3251)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3272..4087)
                     /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     3995..4014
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4088..4190)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(4191..4222)
                     /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 cggtgcctag 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 gccgcgtgcg 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 caccatgacc
     1381 atcgagaagc gctacgactt cgtgttcctg ttcgacgtgc aagacggcaa ccccaacggc
     1441 gatcctgatg ccggaaacct gcctagaatc gaccctcaga caggcgaggg cctcgtgaca
     1501 gatgtgtgcc tgaagcggaa agtgcggaac ttcatccaga tgacccagaa cgacgagcac
     1561 cacgacatct tcatcagaga gaagggcatc ctgaacaacc tgatcgacga ggcccacgag
     1621 caagagaacg tgaagggcaa agagaaaggc gagaaaaccg aggccgccag acagtacatg
     1681 tgcagccggt actacgacat cagaaccttc ggcgccgtga tgaccaccgg caagaatgct
     1741 ggacaagtgc ggggacctgt gcagctgacc ttcagcagat ccatcgatcc catcatgacc
     1801 ctggaacaca gcatcaccag aatggccgtg accaatgaga aggacgccag cgaaaccggc
     1861 gacaacagaa ccatgggcag aaagttcacc gtgccttacg gcctgtaccg gtgccacggc
     1921 tttatcagca cccacttcgc caagcagacc ggcttcagcg agaacgacct ggaactgttt
     1981 tggcaggccc tggtcaacat gttcgatcac gatcactctg ccgccagagg ccagatgaat
     2041 gccagaggac tgtacgtgtt cgagcacagc aacaacctgg gagatgcccc tgccgacagc
     2101 ctgttcaaga gaatccaggt ggtcaagaaa gacggcgtgg aagtcgtgcg gagcttcgac
     2161 gattacctgg tgtccgtgga cgacaagaac ctggaagaga caaagctgct gcggaagctc
     2221 ggcggctctg tgggctatcc ttacgacgtg ccagactacg cccctaagaa aaagcgcaaa
     2281 gtgtgactcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2341 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2401 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2461 ggcaggacag caagggggag gattgggaag acaatagcag gaacgaattc aagcttgata
     2521 tcattcagga cgagcctcag actccagcgt aactggactg caatcaactc actggctcac
     2581 cttcacgggt gggcctttct tcggtagaaa atcaaaggat cttcttgaga tccttttttt
     2641 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2701 ccggatcaag agctaccaac tctttttccg aggtaactgg cttcagcaga gcgcagatac
     2761 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2821 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2881 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2941 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3001 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3061 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3121 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcat cgatttttgt
     3181 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcagaa aggcccaccc
     3241 gaaggtgagc caggtgatta catttgggcc ctcagaaaaa ctcatcgagc atcaagtgaa
     3301 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3361 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3421 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3481 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat
     3541 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3601 caccaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3661 cgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3721 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3781 ctgggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3841 tcggaagagg cataaattcc gtcagccagt ttagcctgac catctcatct gtaacatcat
     3901 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3961 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     4021 aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat
     4081 ggctcatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta
     4141 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca agtcaaaagc
     4201 ctccggtcgg aggcttttga ctttctgcta tggaggtcag gtatgattta aatggtcagt
     4261 attgagcgat atctagagaa ttcgtccgac ggatcggga
//
