LOCUS       sequence-267719-        8389 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Activation of Mafa via SAM system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8389)
  TITLE     Lian Lab CRISPR plasmids
REFERENCE   2  (bases 1 to 8389)
  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"
COMMENT     Sequence Label: pYJ32-lenti-U6-MAFA-acti-gRNA3-MS2
FEATURES             Location/Qualifiers
     source          1..8389
                     /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        2468..2486
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2538..2556
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    2648..2765
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2904..4082
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3135..4073
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4030..4050
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4095..4694
                     /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(4095..4114)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4591..4611
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4710..5298
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4763..4783)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5181..5192)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5370..5603
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5681..5802
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5718..5737)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5772..5791
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5842..5977
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5997..6016)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5998..6016)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6026..6043)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6026..6042)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6035..6057)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6184..6639
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6271..6290)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6481..6502
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6665..6769
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6770..6838,6839..7630)
                     /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(6988..7007)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7801..8389
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8290..8309
                     /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 tctccccccc ctgctccccg gttttagagc
     2461 taggccaaca tgaggatcac ccatgtctgc agggcctagc aagttaaaat aaggctagtc
     2521 cgttatcaac ttggccaaca tgaggatcac ccatgtctgc agggccaagt ggcaccgagt
     2581 cggtgctttt ttggatccaa gcttggcgta actagatctt gagacaaatg gcagtattca
     2641 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     2701 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     2761 attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc gagggggccc
     2821 gggtgcaaag atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct
     2881 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc
     2941 ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt
     3001 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     3061 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     3121 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt
     3181 atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg
     3241 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc
     3301 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc
     3361 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac
     3421 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca
     3481 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat
     3541 gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag
     3601 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg
     3661 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg
     3721 ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca
     3781 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc
     3841 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt
     3901 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta
     3961 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt
     4021 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt
     4081 gacgtacggc caccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     4141 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     4201 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     4261 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcc gtggcggtct
     4321 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     4381 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     4441 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg
     4501 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     4561 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     4621 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     4681 agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa
     4741 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4801 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4861 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4921 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4981 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     5041 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     5101 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     5161 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     5221 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     5281 tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct
     5341 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     5401 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     5461 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     5521 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     5581 gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta ttattcagta
     5641 tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta
     5701 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5761 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct
     5821 atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5881 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     5941 ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct
     6001 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     6061 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     6121 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     6181 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     6241 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     6301 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     6361 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     6421 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     6481 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     6541 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     6601 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     6661 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     6721 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     6781 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     6841 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     6901 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     6961 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     7021 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     7081 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     7141 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     7201 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     7261 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     7321 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     7381 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     7441 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     7501 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     7561 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     7621 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     7681 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     7741 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     7801 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     7861 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     7921 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     7981 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     8041 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     8101 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     8161 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     8221 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     8281 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     8341 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaa
//
