LOCUS       sequence-296670-        7984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for cloning U6 driven LbCpf1 (Cas12a) crRNA, with EF1a 
            driven puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7984)
  AUTHORS   Kempton HR, Goudy LE, Love KS, Qi LS
  TITLE     Multiple Input Sensing and Signal Integration Using a Split Cas12a 
            System.
  JOURNAL   Mol Cell. 2020 Jan 30. pii: S1097-2765(20)30037-X. doi: 
            10.1016/j.molcel.2020.01.016.
  PUBMED    32027839
REFERENCE   2  (bases 1 to 7984)
  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: "Mol Cell. 
            2020 Jan 30. pii: S1097-2765(20)30037-X. doi: 
            10.1016/j.molcel.2020.01.016."
COMMENT     Sequence Label: pSLQ8454 pHR-hU6-LbCpf1 crRNA BB-EF1a-Puro-WPRE
FEATURES             Location/Qualifiers
     source          1..7984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    393..626
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             811..855
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1153..1270
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1329..1577
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1329..1349
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1500..1519
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        1703..1914
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1915..2853
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2810..2830
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2874..3473
                     /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(2874..2893)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3370..3390
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(3493..3526)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3582..4170
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3635..3655)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4053..4064)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4173..4189)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4174..4190)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4699..4879
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4941..5529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5021..5040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5700..6491,6492..6560))
                     /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     6323..6342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6561..6665)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6740..6759)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6931..7310
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7312..7510
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7465..7485
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7528..7708
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7755..7880
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
       61 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
      121 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
      181 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg
      241 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
      301 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
      361 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
      421 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
      481 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
      541 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
      601 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
      661 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
      721 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
      781 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
      841 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
      901 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
      961 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     1021 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga
     1081 tccattcgat tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac aaatggcagt
     1141 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     1201 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     1261 tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggttag taccgggccc
     1321 gctctagaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     1381 ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     1441 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     1501 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     1561 ggaaaggacg aaacaccgaa tttctactaa gtgtagatgg agacgattaa tgcgtctcct
     1621 ttttttaagc ttgggccgct cgagggatcc attaggcggc cgcgtggata accgtattac
     1681 cgccatgcat gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
     1741 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     1801 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     1861 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt
     1921 gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga
     1981 attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
     2041 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
     2101 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
     2161 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
     2221 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
     2281 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
     2341 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
     2401 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
     2461 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
     2521 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
     2581 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
     2641 gattagttct cgcgcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     2701 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     2761 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     2821 acagtggttc aaagtttttt tcttccattt caggtgtcgt gagctagccc accatgaccg
     2881 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc
     2941 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgacccg gaccgccaca
     3001 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     3061 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     3121 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     3181 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     3241 ggttcctggc caccgtcggc gtttctccag accaccaggg caagggtctg ggcagcgccg
     3301 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3361 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3421 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc taagaattcg
     3481 tcgagggacc taataacttc gtatagcata cattatacga agttatacat gtttaagggt
     3541 tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct ctggattaca
     3601 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3661 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3721 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3781 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3841 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3901 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3961 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     4021 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     4081 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     4141 gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta
     4201 gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg
     4261 ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga
     4321 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     4381 gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca
     4441 caaggctact tccctgattg gcagaactac acaccagggc cagggatcag atatccactg
     4501 acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat
     4561 gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag
     4621 agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag
     4681 ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     4741 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     4801 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     4861 gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4921 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4981 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5041 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5101 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5161 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5221 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5281 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5341 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5401 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5461 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5521 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5581 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5641 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5701 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5761 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5821 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5881 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5941 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6001 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6061 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6121 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6181 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6241 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6301 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6361 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6421 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6481 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6541 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6601 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6661 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     6721 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     6781 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     6841 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     6901 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     6961 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     7021 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     7081 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     7141 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     7201 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     7261 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     7321 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     7381 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     7441 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     7501 atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat ctgagcctgg
     7561 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     7621 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     7681 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
     7741 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
     7801 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
     7861 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
     7921 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     7981 ggga
//
