LOCUS       pLenti_mpx_SpCas        9444 bp DNA     circular SYN 17-JUL-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9444)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 14, 2023 from SnapGene Viewer 6.2.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9444
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    2615..2857
                     /label=7SK
     misc_feature    2844..2858
                     /label=3Cs homology
     misc_feature    2858
                     /label=+1
     misc_feature    2859..2878
                     /label=gRNA
     misc_feature    2879..2941
                     /label=spCas9 scaffold V2
     misc_feature    2879..2898
                     /label=3Cs homology
     promoter        3015..3263
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    3248..3264
                     /label=3Cs homology
     misc_feature    3264
                     /label=+1
     misc_feature    3265..3284
                     /label=gRNA
     misc_RNA        3285..3360
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3285..3307
                     /label=3Cs homology
     promoter        3382..3881
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             3921..4520
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    4536..5124
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(5007..5018)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5196..5429
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5461..5685
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5731..6159
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        6173..6502
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6353..6488
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        6550..6597
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6616..6990
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    7120..7241
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7290..7306)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7314..7330
                     /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(7338..7368)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7383..7404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(7692..8280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8451..9311)
                     /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(9312..9416)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttagtac cgggccctac gcgtctgcag tatttagcat gccccaccca
     2641 tctgcaaggc attctggata gtgtcaaaac agccggaaat caagtccgtt tatctcaaac
     2701 tttagcattt tgggaataaa tgatatttgc tatgctggtt aaattagatt ttagttaaat
     2761 ttcctgctga agctctagta cgataagcaa cttgacctaa gtgtaaagtt gagacttcct
     2821 tcaggtttat atagcttgtg cgccgcttgg gtacctcgaa ttaattaatg ttaattaagt
     2881 ttaagagcta tgctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt
     2941 gaaaaagtgg caccgagtcg gtgctttttt tagccgaact gtttcacact cacgcgtcca
     3001 aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca
     3061 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     3121 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     3181 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     3241 tcttgtggaa aggacgaaac accgaatagg gataacaggg taatgtttta gagctagaaa
     3301 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     3361 ttttttggat ccaattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     3421 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     3481 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     3541 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     3601 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
     3661 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     3721 gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg
     3781 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     3841 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcatc catctagatc
     3901 tcgagcagct gaagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     3961 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     4021 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     4081 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     4141 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     4201 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     4261 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc
     4321 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     4381 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     4441 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     4501 cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat tacaaaattt
     4561 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     4621 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     4681 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     4741 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     4801 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     4861 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     4921 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     4981 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     5041 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     5101 tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt
     5161 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     5221 aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg
     5281 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     5341 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     5401 cttttagtca gtgtggaaaa tctctagcag ggcccgttta aacccgctga tcagcctcga
     5461 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     5521 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5581 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5641 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     5701 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     5761 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5821 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5881 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5941 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     6001 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     6061 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     6121 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     6181 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     6241 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     6301 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     6361 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     6421 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     6481 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     6541 tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag
     6601 gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac
     6661 gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag
     6721 gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac
     6781 caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac
     6841 gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc
     6901 gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc
     6961 gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc
     7021 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     7081 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     7141 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     7201 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     7261 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     7321 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     7381 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     7441 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     7501 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     7561 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     7621 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     7681 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     7741 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     7801 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     7861 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     7921 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     7981 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     8041 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     8101 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     8161 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     8221 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     8281 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     8341 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     8401 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     8461 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     8521 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     8581 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     8641 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     8701 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     8761 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     8821 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     8881 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     8941 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     9001 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     9061 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     9121 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     9181 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     9241 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     9301 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     9361 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     9421 tttccccgaa aagtgccacc tgac
//
