LOCUS       sequence-256769-        8388 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Activation of NKX6.1 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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pYJ25-lenti-MS2-U6-NKX6.1-acti-gRNA3
FEATURES             Location/Qualifiers
     source          1..8388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             119..352
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    430..551
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(467..486)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     521..540
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      591..726
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(746..765)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(747..765)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(775..792)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(775..791)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(784..806)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      933..1388
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1020..1039)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1230..1251
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1414..1518
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1519..1587,1588..2379)
                     /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(1737..1756)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2550..3138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3039..3058
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3292..3309
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3426..3447
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3462..3467,3468..3485,3486..3492)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3500..3516
                     /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     3505..3527
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3524..3540
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3524..3540
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3559..3579
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3561..3579
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3607..3833
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             3834..4014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4061..4186
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4679..4912
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5097..5141
                     /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        5319..5567
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5319..5339
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5490..5509
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5605..5623
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        5675..5693
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    5785..5902
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6041..7219
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6272..7210
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7167..7187
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             7232..7831
                     /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(7232..7251)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7728..7748
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    join(7847..8388,1..47)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7900..7920)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8318..8329)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
       61 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
      121 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
      181 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      241 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      301 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat
      361 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
      421 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
      481 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
      541 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
      601 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
      661 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
      721 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
      781 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
      841 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
      901 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
      961 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1021 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1081 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1141 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1201 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1261 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1321 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     1381 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1441 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1501 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1561 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1621 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1681 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     1741 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     1801 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     1861 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     1921 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     1981 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2041 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     2101 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2161 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2221 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2281 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     2341 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2401 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2461 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2521 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2581 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2641 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     2701 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2761 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2821 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2881 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2941 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3001 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3061 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3121 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3181 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3241 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3301 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     3361 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3421 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     3481 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     3541 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa
     3601 gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc
     3661 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt
     3721 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg
     3781 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc
     3841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     3901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     3961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     4021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     4081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     4141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     4201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     4261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     4321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     4381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     4441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     4501 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
     4561 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     4621 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     4681 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     4741 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     4801 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     4861 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     4921 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     4981 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     5041 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     5101 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     5161 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     5221 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     5281 cgtttcagac ccacctccca accccgaggg gacccagaga gggcctattt cccatgattc
     5341 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg
     5401 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     5461 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     5521 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggc gcgagcggag
     5581 aggcactgtt ttagagctag gccaacatga ggatcaccca tgtctgcagg gcctagcaag
     5641 ttaaaataag gctagtccgt tatcaacttg gccaacatga ggatcaccca tgtctgcagg
     5701 gccaagtggc accgagtcgg tgcttttttg gatccaagct tggcgtaact agatcttgag
     5761 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     5821 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     5881 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg
     5941 ccggctcgag ggggcccggg tgcaaagatg gataaagttt taaacagaga ggaatctttg
     6001 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
     6061 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgatc
     6121 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     6181 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     6241 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
     6301 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
     6361 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
     6421 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
     6481 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
     6541 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
     6601 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     6661 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     6721 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     6781 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     6841 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
     6901 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     6961 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     7021 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     7081 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     7141 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     7201 tccatttcag gtgtcgtgac gtacggccac catgaccgag tacaagccca cggtgcgcct
     7261 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     7321 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     7381 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     7441 cgccgccgtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     7501 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     7561 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
     7621 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     7681 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     7741 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     7801 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga
     7861 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     7921 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     7981 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     8041 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     8101 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     8161 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     8221 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     8281 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     8341 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcg
//
