LOCUS       Exported                8922 bp ds-DNA     circular SYN 31-AUG-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pCAG-LbCas12a-Plus-mcherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8922)
  AUTHORS   Triple Threat
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 31, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     ORIGDB|GenBank
FEATURES             Location/Qualifiers
     source          1..8922
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        7..386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        389..664
                     /label=chicken beta-actin promoter
     intron          665..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     promoter        1732..1750
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     promoter        1751..1769
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1771..5454
                     /label=LbCpf1
     misc_feature    2236..2238
                     /label=D156R
     misc_feature    2477..2494
                     /label=LB-724-F
     misc_feature    3296..3317
                     /label=Lb-1525-Fce
     misc_feature    3339..3356
                     /label=LB-1587-F
     primer_bind     complement(3534..3552)
                     /label=Lb-3500-Rce
     misc_feature    4095..4112
                     /label=LB-2343-F
     misc_feature    4417..4431
                     /label=KK/KA
     misc_feature    4417..4419
                     /label=R884K
     misc_feature    4429..4431
                     /label=R888A
     misc_feature    4890..4908
                     /label=Lb-4800-Fce
     CDS             5455..5502
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5509..5589
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             5614..5670
                     /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"
     misc_feature    5630..5648
                     /label=P2A-Fce
     CDS             5677..6387
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     polyA_signal    6430..6551
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     6600..6616
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6624..6640
                     /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(6648..6678)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6693..6714
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(7002..7590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7761..8621)
                     /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"
     promoter        complement(8622..8726)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
       61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      181 tagggacttt ccattgacgt caatgggtgg actatttacg gtaaactgcc cacttggcag
      241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      361 acgtattagt catcgctatt accatgggtc gaggtgagcc ccacgttctg cttcactctc
      421 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      481 gcagcgatgg gggcgggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg
      541 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgttgc cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggctcgtt tcttttctgt ggctgcgtga
      841 aagccttaaa gggctccggg agggcccttt gtgcgggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     1081 gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1261 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccatc tccagcctcg gggctgtccg
     1561 cagggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagtgcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt catttaggtg
     1741 acactataga taatacgact cactataggg atgagcaagc tggagaagtt tacaaactgc
     1801 tactccctgt ctaagaccct gaggttcaag gccatccctg tgggcaagac ccaggagaac
     1861 atcgacaata agcggctgct ggtggaggac gagaagagag ccgaggatta taagggcgtg
     1921 aagaagctgc tggatcgcta ctatctgtct tttatcaacg acgtgctgca cagcatcaag
     1981 ctgaagaatc tgaacaatta catcagcctg ttccggaaga aaaccagaac cgagaaggag
     2041 aataaggagc tggagaacct ggagatcaat ctgcggaagg agatcgccaa ggccttcaag
     2101 ggcaacgagg gctacaagtc cctgtttaag aaggatatca tcgagacaat cctgccagag
     2161 ttcctggacg ataaggacga gatcgccctg gtgaacagct tcaatggctt taccacagcc
     2221 ttcaccggct tctttagaaa cagagagaat atgttttccg aggaggccaa gagcacatcc
     2281 atcgccttca ggtgtatcaa cgagaatctg acccgctaca tctctaatat ggacatcttc
     2341 gagaaggtgg acgccatctt tgataagcac gaggtgcagg agatcaagga gaagatcctg
     2401 aacagcgact atgatgtgga ggatttcttt gagggcgagt tctttaactt tgtgctgaca
     2461 caggagggca tcgacgtgta taacgccatc atcggcggct tcgtgaccga gagcggcgag
     2521 aagatcaagg gcctgaacga gtacatcaac ctgtataatc agaaaaccaa gcagaagctg
     2581 cctaagttta agccactgta taagcaggtg ctgagcgatc gggagtctct gagcttctac
     2641 ggcgagggct atacatccga tgaggaggtg ctggaggtgt ttagaaacac cctgaacaag
     2701 aacagcgaga tcttcagctc catcaagaag ctggagaagc tgttcaagaa ttttgacgag
     2761 tactctagcg ccggcatctt tgtgaagaac ggccccgcca tcagcacaat ctccaaggat
     2821 atcttcggcg agtggaacgt gatccgggac aagtggaatg ccgagtatga cgatatccac
     2881 ctgaagaaga aggccgtggt gaccgagaag tacgaggacg atcggagaaa gtccttcaag
     2941 aagatcggct ccttttctct ggagcagctg caggagtacg ccgacgccga tctgtctgtg
     3001 gtggagaagc tgaaggagat catcatccag aaggtggatg agatctacaa ggtgtatggc
     3061 tcctctgaga agctgttcga cgccgatttt gtgctggaga agagcctgaa gaagaacgac
     3121 gccgtggtgg ccatcatgaa ggacctgctg gattctgtga agagcttcga gaattacatc
     3181 aaggccttct ttggcgaggg caaggagaca aacagggacg agtccttcta tggcgatttt
     3241 gtgctggcct acgacatcct gctgaaggtg gaccacatct acgatgccat ccgcaattat
     3301 gtgacccaga agccctactc taaggataag ttcaagctgt attttcagaa ccctcagttc
     3361 atgggcggct gggacaagga taaggagaca gactatcggg ccaccatcct gagatacggc
     3421 tccaagtact atctggccat catggataag aagtacgcca agtgcctgca gaagatcgac
     3481 aaggacgatg tgaacggcaa ttacgagaag atcaactata agctgctgcc cggccctaat
     3541 aagatgctgc caaaggtgtt cttttctaag aagtggatgg cctactataa ccccagcgag
     3601 gacatccaga agatctacaa gaatggcaca ttcaagaagg gcgatatgtt taacctgaat
     3661 gactgtcaca agctgatcga cttctttaag gatagcatct cccggtatcc aaagtggtcc
     3721 aatgcctacg atttcaactt ttctgagaca gagaagtata aggacatcgc cggcttttac
     3781 agagaggtgg aggagcaggg ctataaggtg agcttcgagt ctgccagcaa gaaggaggtg
     3841 gataagctgg tggaggaggg caagctgtat atgttccaga tctataacaa ggacttttcc
     3901 gataagtctc acggcacacc caatctgcac accatgtact tcaagctgct gtttgacgag
     3961 aacaatcacg gacagatcag gctgagcgga ggagcagagc tgttcatgag gcgcgcctcc
     4021 ctgaagaagg aggagctggt ggtgcaccca gccaactccc ctatcgccaa caagaatcca
     4081 gataatccca agaaaaccac aaccctgtcc tacgacgtgt ataaggataa gaggttttct
     4141 gaggaccagt acgagctgca catcccaatc gccatcaata agtgccccaa gaacatcttc
     4201 aagatcaata cagaggtgcg cgtgctgctg aagcacgacg ataaccccta tgtgatcggc
     4261 atcgataggg gcgagcgcaa tctgctgtat atcgtggtgg tggacggcaa gggcaacatc
     4321 gtggagcagt attccctgaa cgagatcatc aacaacttca acggcatcag gatcaagaca
     4381 gattaccact ctctgctgga caagaaggag aaggagaagt tcgaggccgc ccagaactgg
     4441 acctccatcg agaatatcaa ggagctgaag gccggctata tctctcaggt ggtgcacaag
     4501 atctgcgagc tggtggagaa gtacgatgcc gtgatcgccc tggaggacct gaactctggc
     4561 tttaagaata gccgcgtgaa ggtggagaag caggtgtatc agaagttcga gaagatgctg
     4621 atcgataagc tgaactacat ggtggacaag aagtctaatc cttgtgcaac aggcggcgcc
     4681 ctgaagggct atcagatcac caataagttc gagagcttta agtccatgtc tacccagaac
     4741 ggcttcatct tttacatccc tgcctggctg acatccaaga tcgatccatc taccggcttt
     4801 gtgaacctgc tgaaaaccaa gtataccagc atcgccgatt ccaagaagtt catcagctcc
     4861 tttgacagga tcatgtacgt gcccgaggag gatctgttcg agtttgccct ggactataag
     4921 aacttctctc gcacagacgc cgattacatc aagaagtgga agctgtactc ctacggcaac
     4981 cggatcagaa tcttccggaa tcctaagaag aacaacgtgt tcgactggga ggaggtgtgc
     5041 ctgaccagcg cctataagga gctgttcaac aagtacggca tcaattatca gcagggcgat
     5101 atcagagccc tgctgtgcga gcagtccgac aaggccttct actctagctt tatggccctg
     5161 atgagcctga tgctgcagat gcggaacagc atcacaggcc gcaccgacgt ggattttctg
     5221 atcagccctg tgaagaactc cgacggcatc ttctacgata gccggaacta tgaggcccag
     5281 gagaatgcca tcctgccaaa gaacgccgac gccaatggcg cctataacat cgccagaaag
     5341 gtgctgtggg ccatcggcca gttcaagaag gccgaggacg agaagctgga taaggtgaag
     5401 atcgccatct ctaacaagga gtggctggag tacgcccaga ccagcgtgaa gcacaaaagg
     5461 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agggatccta cccatacgat
     5521 gttccagatt acgcttatcc ctacgacgtg cctgattatg catacccata cgatgtcccc
     5581 gactatgccc tcgagagcac cggtggcagc ggagctacta acttcagcct gctgaagcag
     5641 gctggagacg tggaggagaa ccctggacct gccggtatgg tgagcaaggg cgaggaggat
     5701 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     5761 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     5821 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     5881 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     5941 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     6001 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     6061 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     6121 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     6181 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     6241 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     6301 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     6361 ggcggcatgg acgagctgta caagtaactg cagcgcgggg atctcatgct ggagttcttc
     6421 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     6481 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     6541 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     6601 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     6661 ggaagcataa agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg
     6721 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6781 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     6841 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6901 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6961 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     7021 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     7081 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     7141 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     7201 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     7261 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     7321 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     7381 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     7441 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     7501 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     7561 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7621 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7681 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7741 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7801 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7861 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7921 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7981 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     8041 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     8101 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     8161 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     8221 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     8281 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     8341 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     8401 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     8461 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     8521 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     8581 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8641 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8701 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     8761 ggatcgggag atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat
     8821 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
     8881 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg ac
//
