LOCUS       sequence-296664-        8769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for cloning U6 driven LbCpf1 (Cas12a) crRNA, EF1a driven 
            BFP + puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8769)
  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 8769)
  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: pSLQ8453 pHR-hU6-LbCpf1 crRNA
            BB-EF1a-Puro-t2a...
FEATURES             Location/Qualifiers
     source          1..8769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..118
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        177..425
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     177..197
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     348..367
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        551..762
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          763..1701
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1658..1678
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1736..2332
                     /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(1736..1755)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2232..2252
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2342..2395
                     /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             2408..3106
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(3126..3159)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3215..3803
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3268..3288)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3686..3697)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3806..3822)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3807..3823)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4332..4512
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4574..5162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4654..4673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5333..6124,6125..6193))
                     /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     5956..5975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6194..6298)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6373..6392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6564..6943
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6945..7143
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7098..7118
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7161..7341
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7388..7513
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8010..8243
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8428..8472
                     /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"
ORIGIN
        1 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
       61 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
      121 ggtttattac agggacagca gagatccagt ttggttagta ccgggcccgc tctagagagg
      181 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
      241 ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
      301 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
      361 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
      421 acaccgaatt tctactaagt gtagatggag acgattaatg cgtctccttt ttttaagctt
      481 gggccgctcg agggatccat taggcggccg cgtggataac cgtattaccg ccatgcatgt
      541 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      601 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      661 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      721 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
      781 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
      841 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
      901 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
      961 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     1021 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     1081 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
     1141 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     1201 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     1261 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     1321 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     1381 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     1441 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1501 cgcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1561 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1621 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1681 agtttttttc ttccatttca ggtgtcgtga gctagcccac catgaccgac ccaccatgac
     1741 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac
     1801 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca
     1861 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     1921 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     1981 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     2041 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     2101 gtggttcctg gccaccgtcg gcgtttctcc agaccaccag ggcaagggtc tgggcagcgc
     2161 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     2221 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     2281 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccggatcggg
     2341 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaccggt
     2401 cgccaccagc gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt
     2461 ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
     2521 gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
     2581 tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt
     2641 cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg
     2701 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
     2761 caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg
     2821 ctgggaggcc ttcaccgaga ctctgtatcc tgctgacggc ggcctggaag gcagaaacga
     2881 catggccctg aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag
     2941 atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact
     3001 ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc
     3061 cagatactgc gacctcccta gcaaactggg gcacaagctt aattaagaat tcgtcgaggg
     3121 acctaataac ttcgtatagc atacattata cgaagttata catgtttaag ggttccggtt
     3181 ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
     3241 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3301 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3361 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3421 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3481 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3541 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3601 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     3661 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3721 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3781 ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac
     3841 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag
     3901 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga
     3961 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     4021 taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct
     4081 acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg
     4141 gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag
     4201 agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag
     4261 tattagagtg gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc
     4321 cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     4381 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     4441 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     4501 aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4561 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4621 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4681 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4741 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4801 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4861 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4921 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4981 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5041 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5101 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5161 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5221 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5281 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5341 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5401 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5461 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5521 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5581 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5641 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5701 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5761 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5821 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5881 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5941 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6001 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6061 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6121 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6181 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6241 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6301 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     6361 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6421 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     6481 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     6541 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     6601 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     6661 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     6721 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     6781 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     6841 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     6901 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     6961 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     7021 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     7081 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     7141 cagcgcgttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     7201 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     7261 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     7321 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     7381 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     7441 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     7501 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     7561 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     7621 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     7681 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     7741 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     7801 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     7861 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     7921 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     7981 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     8041 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     8101 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     8161 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     8221 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     8281 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     8341 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     8401 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     8461 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     8521 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     8581 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     8641 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     8701 attcgattag tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt
     8761 catccacaa
//
