LOCUS       sequence-256658-        8388 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Activation of Mafa via SAM system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8388)
  TITLE     Lian Lab CRISPR plasmids
REFERENCE   2  (bases 1 to 8388)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pYJ31-lenti-U6-MAFA-acti-gRNA2-MS2
FEATURES             Location/Qualifiers
     source          1..8388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    288..309
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(324..329,330..347,348..354)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    362..378
                     /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)."
     primer_bind     367..389
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     386..402
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     386..402
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     421..441
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        423..441
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        469..695
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             696..876
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    923..1048
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1541..1774
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1959..2003
                     /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"
     promoter        2181..2429
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2181..2201
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2352..2371
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2467..2485
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2537..2555
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    2647..2764
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2903..4081
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3134..4072
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4029..4049
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4094..4693
                     /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(4094..4113)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4590..4610
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4709..5297
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4762..4782)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5180..5191)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5369..5602
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5680..5801
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5717..5736)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5771..5790
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5841..5976
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5996..6015)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5997..6015)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6025..6042)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6025..6041)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6034..6056)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6183..6638
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6270..6289)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6480..6501
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6664..6768
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6769..6837,6838..7629)
                     /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     complement(6987..7006)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7800..8388
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8289..8308
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      241 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      301 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      361 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
      421 gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg
      481 caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag
      541 agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag
      601 gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag
      661 atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct
      721 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
      781 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
      841 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
      901 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
      961 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     1021 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     1081 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     1141 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     1201 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1261 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1321 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1381 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     1441 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     1501 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     1561 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     1621 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     1681 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     1741 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     1801 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     1861 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     1921 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     1981 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     2041 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     2101 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     2161 caaccccgag gggacccaga gagggcctat ttcccatgat tccttcatat ttgcatatac
     2221 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
     2281 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2341 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2401 tatatatctt gtggaaagga cgaaacaccg gcggcggtcc cggcgggggg ttttagagct
     2461 aggccaacat gaggatcacc catgtctgca gggcctagca agttaaaata aggctagtcc
     2521 gttatcaact tggccaacat gaggatcacc catgtctgca gggccaagtg gcaccgagtc
     2581 ggtgcttttt tggatccaag cttggcgtaa ctagatcttg agacaaatgg cagtattcat
     2641 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     2701 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     2761 ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg agggggcccg
     2821 ggtgcaaaga tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta
     2881 ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc
     2941 cacagtcccc gagaagttgg ggggaggggt cggcaattga tccggtgcct agagaaggtg
     3001 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     3061 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     3121 cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta
     3181 tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga
     3241 gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc
     3301 tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca
     3361 ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc
     3421 tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac
     3481 tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg
     3541 ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc
     3601 tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc
     3661 aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc
     3721 tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac
     3781 acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg
     3841 ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg
     3901 gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag
     3961 gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg
     4021 gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg
     4081 acgtacggcc accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     4141 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     4201 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     4261 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgccg tggcggtctg
     4321 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     4381 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     4441 ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg
     4501 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     4561 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     4621 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     4681 gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag
     4741 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     4801 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     4861 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     4921 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     4981 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     5041 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     5101 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     5161 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     5221 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     5281 ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt
     5341 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     5401 gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     5461 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     5521 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     5581 tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat
     5641 ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat
     5701 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5761 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta
     5821 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     5881 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     5941 tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta
     6001 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     6061 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     6121 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     6181 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6241 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6301 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     6361 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     6421 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     6481 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     6541 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     6601 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     6661 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     6721 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     6781 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     6841 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     6901 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     6961 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     7021 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     7081 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     7141 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     7201 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     7261 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     7321 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     7381 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     7441 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     7501 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     7561 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     7621 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     7681 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     7741 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     7801 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     7861 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     7921 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     7981 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     8041 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     8101 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     8161 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     8221 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     8281 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     8341 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaa
//
