LOCUS       sequence-351754-        5970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human codon-optimized NlaCas3 with C terminal NLS and 
            HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5970)
  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 5970)
  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-cas3
FEATURES             Location/Qualifiers
     source          1..5970
                     /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             3889..3915
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3934..3954
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3970..3987)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3976..4087
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     terminator      4245..4274
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      4296..4883
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4784..4803
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4895..4922)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4943..5758)
                     /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     5666..5685
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5759..5861)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(5862..5893)
                     /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 caccatgaac
     1381 ttcgactata tcgcccacgc cagacaggac agcagcaaga actggcactc tcaccctctg
     1441 cagaaacatc tgcagaaggt ggcccagctg gccaagagat ttgccggcag atacggcagc
     1501 ctgttcgccg aatatgccgg cctgctgcac gatctgggca agttccaaga gagcttccag
     1561 aagtacatcc ggaacgccag cggcttcgag aaagagaatg cccacctgga agatgtggaa
     1621 agcaccaagc tgcggaagat ccctcactct acagccggcg ctaagtacgc cgtggaaaga
     1681 ctgaacccct tcttcggcca tctgctggcc tatctgattg ccggacatca tgccggactg
     1741 gccgattggt acgataaggg cagcctgaag cggagactgc agcaagccga tgatgaactg
     1801 gccgcctctc tgtccggctt cgtggaatct tctctgcccg aggacttctt ccctctgtcc
     1861 gacgacgacc tgatgagaga cttcttcgcc ttctgggagg acggcgccaa gctggaagaa
     1921 ctgcacatct ggatgcggtt tctgttcagc tgcctggtgg acgccgactt cctggatacc
     1981 gaggccttca tgaacggcta cgccgatgcc gatacagccc aagctgctgg actgaggcct
     2041 aagttccctg gcctggatga gctgcatcgg agatacgagc agtacatggc tcagctgtcc
     2101 gagaaggccg acaagaacag ctccctgaat caagagcggc acgccatcct gcagcagtgc
     2161 ttttctgccg ccgagacaga cagaaccctg ttcagcctga cagtgcctac aggcggcgga
     2221 aaaactctgg cctctctggg ctttgccctg aagcacgccc tgaagttcgg caagaagcgg
     2281 atcatctacg ccattccttt caccagcatc atcgagcaga acgccaacgt gttcagaaac
     2341 gccctgggcg acgatgtggt gctggaacac cacagcaacc tggaagtgaa agaggacaaa
     2401 gagacagcca agaccagact ggccaccgag aattgggatg cccctctgat cgtgaccacc
     2461 aacgtgcagc tgttcgagag cctgtttgcc gccaagacct ccagatgcag aaagatccac
     2521 aatatcgccg acagcgtggt catcctggac gaagctcagc agctgccccg ggacttccag
     2581 aaacctatca ccgatatgat gcgcgtgctg gccagagact acggcgtgac ctttgtgctg
     2641 tgtaccgcca cacagcctga gctgggcaag aacatcgatg ccttcggccg gaccatcctg
     2701 gaaggattgc ctgacgtgcg ggaaatcgtg gccgataaga tcgccctgag cgagaagctg
     2761 agaagagtgc ggatcaagat gcctcctcca aacggcgaga cacagagctg gcagaagatc
     2821 gccgacgaga tcgccgctag accatgtgtg ctggccgtgg tcaacaccag aaaacacgcc
     2881 cagaagctgt tcgctgccct gcctagcaat ggcatcaagc tgcacctgag cgccaacatg
     2941 tgcgccacac actgctctga agtgatcgcc ctcgtgcgga gatatctggc cctgtacaga
     3001 gccggaagcc tgcacaaacc tctgtggctg gtgtctaccc agctgattga agctggcgtg
     3061 gacctggact tcccctgtgt gtatagagcc atggccggcc tggattctat tgcccaagca
     3121 gccggacggt gcaacagaga gggaaaactg cctcagctgg gcgaagtggt ggtgttcaga
     3181 gctgaagaag gcgcccctag cggctctctg aagcaaggcc aggatatcac cgaggaaatg
     3241 ctgaaggccg gactgctgga cgaccctttg tctcctctgg ccttcgccga gtacttcaga
     3301 cggttcaatg gcaagggcga cgtggacaag cacggcatca caacactgct gacagccgag
     3361 gccagcaacg agaatccact ggccatcaag ttccggaccg ccgctgagag attccacctg
     3421 atcgataatc agggcgtcgc actgatcgtg cccttcattc ctctggctca ctgggagaaa
     3481 gacggcagcc ctcagatcgt ggaagccaac gagctggacg atttcttcag gcggcacctg
     3541 gacggcgtgg aagtgtctga gtggcaggac atcctggata agcagcggtt ccctcagcct
     3601 cctgacaaca gctttggcca gaccgatcag cctctgctgc ctgagccttt cgagagttgg
     3661 ttcggcctgc tcgagagcga cccactgaag cacaaatggg tgtaccggaa gctgcagcgg
     3721 tacaccatca ccgtgtatga gcacgagctg aaaaagctgc ccgagcacgc cgtgttctct
     3781 agagctggac tgctcgtgct ggacaagggc tactataagg ccgtgctggg cgccgatttt
     3841 gacgatgctg cttggctgcc agagaactct gtgctgggct ctgtgggcta cccctacgat
     3901 gtgcctgatt acgccggcag ctaccctgag ttccccaaga aaaagcggaa agtgtgactc
     3961 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     4021 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     4081 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     4141 gcaaggggga ggattgggaa gacaatagca ggaacgaatt caagcttgat atcattcagg
     4201 acgagcctca gactccagcg taactggact gcaatcaact cactggctca ccttcacggg
     4261 tgggcctttc ttcggtagaa aatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4321 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4381 gagctaccaa ctctttttcc gaggtaactg gcttcagcag agcgcagata ccaaatactg
     4441 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4501 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4561 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4621 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4681 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4741 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4801 tttatagtcc tgtcgggttt cgccacctct gacttgagca tcgatttttg tgatgctcgt
     4861 caggggggcg gagcctatgg aaaaacgcca gcaacgcaga aaggcccacc cgaaggtgag
     4921 ccaggtgatt acatttgggc cctcagaaaa actcatcgag catcaagtga aactgcaatt
     4981 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     5041 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     5101 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     5161 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta tgcatttctt
     5221 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcaccaacca
     5281 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ccgttaaaag
     5341 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     5401 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc cctgggatcg
     5461 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     5521 gcataaattc cgtcagccag tttagcctga ccatctcatc tgtaacatca ttggcaacgc
     5581 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     5641 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     5701 ccatgttgga atttaatcgc ggcctcgagc aagacgtttc ccgttgaata tggctcattt
     5761 tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta
     5821 tttcattatg gtgaaagttg gaacctctta cgtgccgatc aagtcaaaag cctccggtcg
     5881 gaggcttttg actttctgct atggaggtca ggtatgattt aaatggtcag tattgagcga
     5941 tatctagaga attcgtccga cggatcggga
//
