LOCUS       sequence-351750-        5208 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human codon-optimized NlaCas8 with N terminal NLS and 
            HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5208)
  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 5208)
  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-cas8
FEATURES             Location/Qualifiers
     source          1..5208
                     /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"
     regulatory      1369..1378
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1381..1401
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1402..1428
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3208..3225)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3214..3325
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     terminator      3483..3512
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      3534..4121
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4022..4041
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4133..4160)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4181..4996)
                     /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     4904..4923
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4997..5099)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(5100..5131)
                     /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 caccatggtg
     1381 cccaagaaaa agcggaaggt gtacccctac gacgtgcccg attatgccgg ctctgtggga
     1441 attctgcacg ccctgacaca gtactaccag cggaaggccg aaagcgacgg cggaattgcc
     1501 caagagggct tcgagaacaa agagatcccc ttcatcatcg tgatcgacaa gcagggcaac
     1561 ttcatccagc tcgaggacac ccgcgagctg aaagtgaaga aaaaagtggg ccgcaccttc
     1621 ctggtgccta aaggccttgg cagaagcggc agcaagagct acgaggtgtc caacctgctg
     1681 tgggaccact acggatacgt gctggcctat gccggcgaga agggacaaga acaggccgat
     1741 aagcagcacg ccagcttcac cgccaaagtg aacgagctga agcaggccct gcctgatgat
     1801 gctggcgtga cagctgtggc cgcctttctg tctagcgccg aagagaagtc caaagtgatg
     1861 caggccgcca actgggccga gtgcgctaaa gtgaagggct gcaacctgag cttccggctg
     1921 gtggatgaag ccgtggatct cgtgtgtcag tctaaggccg tgcgcgagta tgtgtcccag
     1981 gccaatcaga cccagagcga caacgtgcag aaaggcatct gtctggtcac cggcaaggcc
     2041 gctcctattg ccagactgca caatgccgtg aagggcgtga acgccaagcc tgctcctttc
     2101 gcctctgtga acctgagcgc ctttgagagc tacggcaaag agcagggctt catcttccct
     2161 gtgggagagc aggccatgtt cgagtacacc accgctctga ataccctgct ggcctccgag
     2221 aacagattcc ggatcggaga tgtgaccgcc gtgtgttggg gagccaagag aacacctctg
     2281 gaagagtccc tggccagcat gatcaatggc ggcggaaagg acaagcccga cgagcacatc
     2341 gacgccgtga aaaccctgta caagagcctg tacaacggcc agtaccagaa gcctgacgga
     2401 aaagagaagt tctacctgct gggactgagc cccaacagcg ccagaatcgt tgtgcggttc
     2461 tggcacgaga caaccgtggc tgccctgtct gagtctatcg ccgcttggta cgacgacctg
     2521 cagatggttc gaggcgagaa cagcccctat cctgagtaca tgcccctgcc tagactgctg
     2581 ggcaacctgg tgctggacgg caagatggaa aacctgccta gcgacctgat cgcccagatc
     2641 acagatgctg ccctgaacaa cagagtgctg cctgtcagtc tgctgcaggc agccctgaga
     2701 agaaacaagg ccgagcagaa gatcacctac ggcagagcca gcctgctgaa ggcctacatc
     2761 aaccgggcca tcagagccgg acggctgaag aacatgaagg aactgaccat gggcctcgac
     2821 cggaacagac aggatatcgg ctatgtgctg ggcagactgt tcgccgtgct ggaaaagatt
     2881 caggccgagg ccaatcctgg cctgaacgcc acaatcgccg acagatattt tggcagcgcc
     2941 agcagcacac ctatcgccgt gtttggcacc ctgatgagac tgctgcctca ccacctgaac
     3001 aagctggaat tcgagggcag agccgtgcag ctccagtggg agatcagaca gatcctggaa
     3061 cactgccagc ggttccccaa tcacctgaac ctggaacagc agggactgtt tgccatcggc
     3121 tactaccacg agacacagtt tctgttcacc aaggacgccc tgaagaacct gttcaacgag
     3181 gccaagaccg cctgactcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3241 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3301 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3361 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg aacgaattca
     3421 agcttgatat cattcaggac gagcctcaga ctccagcgta actggactgc aatcaactca
     3481 ctggctcacc ttcacgggtg ggcctttctt cggtagaaaa tcaaaggatc ttcttgagat
     3541 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3601 gtttgtttgc cggatcaaga gctaccaact ctttttccga ggtaactggc ttcagcagag
     3661 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3901 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3961 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4021 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcatc
     4081 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcagaaa
     4141 ggcccacccg aaggtgagcc aggtgattac atttgggccc tcagaaaaac tcatcgagca
     4201 tcaagtgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     4261 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     4321 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     4381 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     4441 aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     4501 aatcactcgc accaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     4561 cgcgatcgcc gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     4621 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     4681 ctgttttccc tgggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     4741 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagcctgacc atctcatctg
     4801 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     4861 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     4921 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc
     4981 gttgaatatg gctcatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
     5041 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     5101 gtcaaaagcc tccggtcgga ggcttttgac tttctgctat ggaggtcagg tatgatttaa
     5161 atggtcagta ttgagcgata tctagagaat tcgtccgacg gatcggga
//
