LOCUS       sequence-335749-        5580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac Cas13d sgRNA plasmid for CLCN3 knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5580)
  AUTHORS   Chang Y, Syahirah R, Wang X, Jin G, Torregrosa-Allen S, Elzey BD, 
            Hummel SN, Wang T, Li C, Lian X, Deng Q, Broxmeyer HE, Bao X
  TITLE     Engineering chimeric antigen receptor neutrophils from human 
            pluripotent stem cells for targeted cancer immunotherapy.
  JOURNAL   Cell Rep. 2022 Jul 19;40(3):111128. doi: 
            10.1016/j.celrep.2022.111128.
  PUBMED    35858579
REFERENCE   2  (bases 1 to 5580)
  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"; journalName: "Cell Rep.";
            date: "2022-07-19"; volume: "40(3):111128. doi"; pages: " 
            10.1016/j.celrep.2022.111128"
COMMENT     Sequence Label: PiggyBac EF1a-eGFP-Puro U6 CLCN3 g1
FEATURES             Location/Qualifiers
     source          1..5580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..212
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      232..241
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(238..240,241..243,244..954)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(238..240,241..243,244..954)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(283..304)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(544..563)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     891..912
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             955..975
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             991..1044
                     /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             1054..1653
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1054..1073)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1550..1570
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1674..1755
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1756..2004
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1756..1776
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1927..1946
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2006..2035
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     repeat_region   2241..2303
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2329..2345)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2329..2345)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2342..2364)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2353..2369
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2377..2383,2384..2401,2402..2407))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2422..2443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2560..2577)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2731..3319)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2811..2830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3490..4281,4282..4350))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4113..4132
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4351..4455)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4523..4541
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4579..4601)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4701..4720
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4914..4936
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4928..4945
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4929..4945
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   4956..4990
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5266..5400
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
       61 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      121 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      181 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc cgccaccatg
      241 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      301 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      361 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      421 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      481 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      541 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      601 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      661 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
      721 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
      781 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      841 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
      901 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagccaaag
      961 aagaagcgta aggtacccgg gggcagcgga gagggcagag gttctcttct aacatgcggt
     1021 gacgtggagg agaatcccgg ccctaggctc gagatgaccg agtacaagcc cacggtgcgc
     1081 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     1141 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     1201 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     1261 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     1321 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     1381 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     1441 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     1501 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     1561 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     1621 acctggtgca tgacccgcaa gcccggtgcc tgatctagag ggcccgttta gttaacttgt
     1681 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     1741 catttttttc actgcgaggg cctatttccc atgattcctt catatttgca tatacgatac
     1801 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     1861 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     1921 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     1981 atcttgtgga aaggacgaaa caccgaaccc ctaccaactg gtcggggttt gaaacgaaaa
     2041 gatatttccg cagcaacatt ttttttaaaa gttttgttac tttatagaag aaattttgag
     2101 tttttgtttt tttttaataa ataaataaac ataaataaat tgtttgttga atttattatt
     2161 agtatgtaag tgtaaatata ataaaactta atatctattc aaattaataa ataaacctcg
     2221 atatacagac cgataaaaca catgcgtcaa ttttacgcat gattatcttt aacgtacgtc
     2281 acaatatgat tatctttcta gggttaagtc gaccttggcg taatcatggt catagctgtt
     2341 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2401 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2461 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2521 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     2581 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2641 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2701 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2761 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2821 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2881 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     2941 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3001 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3061 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3121 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3181 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3241 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3301 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3361 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3421 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3481 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3541 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3601 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3661 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3721 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3781 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3841 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3901 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3961 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4021 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4081 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4141 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4201 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4261 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4321 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4381 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4441 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     4501 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     4561 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     4621 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4681 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     4741 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
     4801 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     4861 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     4921 acgacgttgt aaaacgacgg ccagtgaatt cttaacccta gaaagatagt ctgcgtaaaa
     4981 ttgacgcatg cattcttgaa atattgctct ctctttctaa atagcgcgaa tccgtcgctg
     5041 tgcatttagg acatctcagt cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg
     5101 taagtgtcac tgattttgaa ctataacgac cgcgtgagtc aaaatgacgc atgattatct
     5161 tttacgtgac ttttaagatt taactcatac gataattata ttgttatttc atgttctact
     5221 tacgtgataa cttattatat atatattttc ttgttataga tatcaaactt gtttattgca
     5281 gcttataatg gttacaaata aggcaatagc atcacaaatt tcacaaataa ggcatttttt
     5341 tcactgcatt ctagttttgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     5401 tcaaatccct cggaagctgc gcctgtctta ggttggagtg atacattttt atcactttta
     5461 cccgtctttg gattaggcag tagctctgac ggccctcctg tcttaggtta gtgaaaaatg
     5521 tcactctctt acccgtcatt ggctgtccag cttagctcgc aggggaggtg gtctactagt
//
