LOCUS       sequence-383727-        7287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  human codon optimized Nla-cas8c-Cas7c-Cas5c-Cas11c cloned into 
            pSmart-HC-Kan-EF1a-bGH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7287)
  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 7287)
  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-Kan-HC-EF1a-Nla-Cas8-7-5-11
FEATURES             Location/Qualifiers
     source          1..7287
                     /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             3136..3189
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4054..4080
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4081..4101
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4111..4167
                     /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"
     CDS             4825..4884
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     primer_bind     complement(5287..5304)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5293..5404
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     terminator      5562..5591
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      5613..6200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6101..6120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6212..6239)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(6260..7075)
                     /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     6983..7002
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(7076..7178)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(7179..7210)
                     /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 caccatgatt
     1381 ctgcacgccc tgacacagta ctaccagcgg aaggccgaaa gcgacggcgg aattgcccaa
     1441 gagggcttcg agaacaaaga gatccccttc atcatcgtga tcgacaagca gggcaacttc
     1501 atccagctcg aggacacccg cgagctgaaa gtgaagaaaa aagtgggccg caccttcctg
     1561 gtgcctaaag gccttggcag aagcggcagc aagagctacg aggtgtccaa cctgctgtgg
     1621 gaccactacg gatacgtgct ggcctatgcc ggcgagaagg gacaagaaca ggccgataag
     1681 cagcacgcca gcttcaccgc caaagtgaac gagctgaagc aggccctgcc tgatgatgct
     1741 ggcgtgacag ctgtggccgc ctttctgtct agcgccgaag agaagtccaa agtgatgcag
     1801 gccgccaact gggccgagtg cgctaaagtg aagggctgca acctgagctt ccggctggtg
     1861 gatgaagccg tggatctcgt gtgtcagtct aaggccgtgc gcgagtatgt gtcccaggcc
     1921 aatcagaccc agagcgacaa cgtgcagaaa ggcatctgtc tggtcaccgg caaggccgct
     1981 cctattgcca gactgcacaa tgccgtgaag ggcgtgaacg ccaagcctgc tcctttcgcc
     2041 tctgtgaacc tgagcgcctt tgagagctac ggcaaagagc agggcttcat cttccctgtg
     2101 ggagagcagg ccatgttcga gtacaccacc gctctgaata ccctgctggc ctccgagaac
     2161 agattccgga tcggagatgt gaccgccgtg tgttggggag ccaagagaac acctctggaa
     2221 gagtccctgg ccagcatgat caatggcggc ggaaaggaca agcccgacga gcacatcgac
     2281 gccgtgaaaa ccctgtacaa gagcctgtac aacggccagt accagaagcc tgacggaaaa
     2341 gagaagttct acctgctggg actgagcccc aacagcgcca gaatcgttgt gcggttctgg
     2401 cacgagacaa ccgtggctgc cctgtctgag tctatcgccg cttggtacga cgacctgcag
     2461 atggttcgag gcgagaacag cccctatcct gagtacatgc ccctgcctag actgctgggc
     2521 aacctggtgc tggacggcaa gatggaaaac ctgcctagcg acctgatcgc ccagatcaca
     2581 gatgctgccc tgaacaacag agtgctgcct gtcagtctgc tgcaggcagc cctgagaaga
     2641 aacaaggccg agcagaagat cacctacggc agagccagcc tgctgaaggc ctacatcaac
     2701 cgggccatca gagccggacg gctgaagaac atgaaggaac tgaccatggg cctcgaccgg
     2761 aacagacagg atatcggcta tgtgctgggc agactgttcg ccgtgctgga aaagattcag
     2821 gccgaggcca atcctggcct gaacgccaca atcgccgaca gatattttgg cagcgccagc
     2881 agcacaccta tcgccgtgtt tggcaccctg atgagactgc tgcctcacca cctgaacaag
     2941 ctggaattcg agggcagagc cgtgcagctc cagtgggaga tcagacagat cctggaacac
     3001 tgccagcggt tccccaatca cctgaacctg gaacagcagg gactgtttgc catcggctac
     3061 taccacgaga cacagtttct gttcaccaag gacgccctga agaacctgtt caacgaggcc
     3121 aagaccgccg gctccgaggg ccggggctct ctgctgacct gtggcgacgt ggaggagaac
     3181 cccggcccct ccatgaccat cgagaagcgc tacgacttcg tgttcctgtt cgacgtgcaa
     3241 gacggcaacc ccaacggcga tcctgatgcc ggaaacctgc ctagaatcga ccctcagaca
     3301 ggcgagggcc tcgtgacaga tgtgtgcctg aagcggaaag tgcggaactt catccagatg
     3361 acccagaacg acgagcacca cgacatcttc atcagagaga agggcatcct gaacaacctg
     3421 atcgacgagg cccacgagca agagaacgtg aagggcaaag agaaaggcga gaaaaccgag
     3481 gccgccagac agtacatgtg cagccggtac tacgacatca gaaccttcgg cgccgtgatg
     3541 accaccggca agaatgctgg acaagtgcgg ggacctgtgc agctgacctt cagcagatcc
     3601 atcgatccca tcatgaccct ggaacacagc atcaccagaa tggccgtgac caatgagaag
     3661 gacgccagcg aaaccggcga caacagaacc atgggcagaa agttcaccgt gccttacggc
     3721 ctgtaccggt gccacggctt tatcagcacc cacttcgcca agcagaccgg cttcagcgag
     3781 aacgacctgg aactgttttg gcaggccctg gtcaacatgt tcgatcacga tcactctgcc
     3841 gccagaggcc agatgaatgc cagaggactg tacgtgttcg agcacagcaa caacctggga
     3901 gatgcccctg ccgacagcct gttcaagaga atccaggtgg tcaagaaaga cggcgtggaa
     3961 gtcgtgcgga gcttcgacga ttacctggtg tccgtggacg acaagaacct ggaagagaca
     4021 aagctgctgc ggaagctcgg cggctctgtg ggctatcctt acgacgtgcc agactacgcc
     4081 cctaagaaaa agcggaaagt gggctccgga gccaccaact tctccctgct gaagcaggcc
     4141 ggcgacgtgg aggagaaccc cggcccctcc atgcggttca tcctggaaat cagcggcgac
     4201 ctggcctgct tcacaagaag cgagctgaag gtcgagcggg tgtcataccc tgtgatcacc
     4261 cctagcgccg ccagaaacat cctgatggcc attctgtgga agcccgccat cagatggaag
     4321 gtgctgaaga tcgagatcct gaagcctatc cagtggacca acatccggcg gaacgaagtg
     4381 ggcaccaaga tgagcgagag aagcggcagc ctgtacatcg aggacaacag acagcagcgg
     4441 gcctccatgc tgctgaagga tgtggcctat agaatccacg ccgacttcga catgacaagc
     4501 gaggccggcg agagcgacaa ctacgtgaag ttcgccgaga tgttcaagcg gagagccaag
     4561 aagggccagt acttccacca gccttacctg ggctgcagag agttcccctg cgacttcaga
     4621 ctgctggaaa aggccgagga tggcctgcct ctggaagata tcacccagga cttcggcttc
     4681 atgctgtacg acatggactt cagcaagagc gaccccagag acagcaacaa cgccgagcct
     4741 atgttctacc agtgcaaggc cgtgaacggc gtgatcactg tgcctccagc cgatagcgag
     4801 gaagtgaaga gaggctctgt gggccagtgc acaaactacg ccctgttgaa gctggctgga
     4861 gacgtggagt ccaaccctgg accctccatg ggcctcgacc ggaacagaca ggatatcggc
     4921 tatgtgctgg gcagactgtt cgccgtgctg gaaaagattc aggccgaggc caatcctggc
     4981 ctgaacgcca caatcgccga cagatatttt ggcagcgcca gcagcacacc tatcgccgtg
     5041 tttggcaccc tgatgagact gctgcctcac cacctgaaca agctggaatt cgagggcaga
     5101 gccgtgcagc tccagtggga gatcagacag atcctggaac actgccagcg gttccccaat
     5161 cacctgaacc tggaacagca gggactgttt gccatcggct actaccacga gacacagttt
     5221 ctgttcacca aggacgccct gaagaacctg ttcaacgagg ccaagaccgc ctgactcgct
     5281 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     5341 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     5401 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     5461 agggggagga ttgggaagac aatagcagga acgaattcaa gcttgatatc attcaggacg
     5521 agcctcagac tccagcgtaa ctggactgca atcaactcac tggctcacct tcacgggtgg
     5581 gcctttcttc ggtagaaaat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5641 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5701 ctaccaactc tttttccgag gtaactggct tcagcagagc gcagatacca aatactgttc
     5761 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5821 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5881 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5941 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6001 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6061 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6121 atagtcctgt cgggtttcgc cacctctgac ttgagcatcg atttttgtga tgctcgtcag
     6181 gggggcggag cctatggaaa aacgccagca acgcagaaag gcccacccga aggtgagcca
     6241 ggtgattaca tttgggccct cagaaaaact catcgagcat caagtgaaac tgcaatttat
     6301 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     6361 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     6421 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     6481 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc
     6541 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca ccaaccaaac
     6601 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgccg ttaaaaggac
     6661 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     6721 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttccct gggatcgcag
     6781 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     6841 taaattccgt cagccagttt agcctgacca tctcatctgt aacatcattg gcaacgctac
     6901 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     6961 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     7021 tgttggaatt taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcattttag
     7081 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt
     7141 cattatggtg aaagttggaa cctcttacgt gccgatcaag tcaaaagcct ccggtcggag
     7201 gcttttgact ttctgctatg gaggtcaggt atgatttaaa tggtcagtat tgagcgatat
     7261 ctagagaatt cgtccgacgg atcggga
//
