LOCUS       sequence-256775-        4134 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 4134)
  TITLE     Lian Lab CRISPR plasmids
REFERENCE   2  (bases 1 to 4134)
  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: pYJ15-PB-U6-Nkx6.1-acti-gRNA1
FEATURES             Location/Qualifiers
     source          1..4134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     173..191
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(229..251)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     351..370
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        451..469
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     451..468
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    535..556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(571..576,577..594,595..601)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    609..625
                     /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     614..636
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     633..649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     633..649
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        670..688
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   871..933
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1227..1475
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1227..1247
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1398..1417
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1514..1532
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1584..1602
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     repeat_region   2080..2114
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2238..2257)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2239..2257)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2344..2361)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2515..3103)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2595..2614)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3274..4065,4066..4134))
                     /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     3897..3916
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
      181 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      241 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      301 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      361 gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta
      421 atacataacc ttatgtatca tacacatacg atttaggtga cactatagaa ctcgacctcg
      481 aggctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      541 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      601 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      661 caagctcgaa attaaccctc actaaaggga acaaaagctg gagctcgtct ttgatcaaaa
      721 cgcaaatcga cgaaaatgtg tcggacaata tcaagtcgat gagcgaaaaa ctaaaaaggc
      781 tagaatacga caatctcaca gacagcgttg agatatacgg tattcacgac agcaggctga
      841 ataataaaaa aattagaaac tattatttaa ccctagaaag ataatcatat tgtgacgtac
      901 gttaaagata atcatgcgta aaattgacgc atgtgtttta tcgatctgta tatcgaggtt
      961 tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata
     1021 aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa
     1081 tttcttctat aaagtaacaa aacttttaaa cattctctct tttacaaaaa taaacttatt
     1141 ttgtacttta aaaacagtca tgttgtatta taaaataagt aattagctta acttatacat
     1201 aatagaaaca aattatactt attaatgagg gcctatttcc catgattcct tcatatttgc
     1261 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     1321 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     1381 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     1441 tggctttata tatcttgtgg aaaggacgaa acaccgcaga ggagccggaa gcgccggttt
     1501 tagagctagg ccaacatgag gatcacccat gtctgcaggg cctagcaagt taaaataagg
     1561 ctagtccgtt atcaacttgg ccaacatgag gatcacccat gtctgcaggg ccaagtggca
     1621 ccgagtcggt gctttttttg aattctgatg cggtattttc tccttacgca tctgtgcggt
     1681 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc atttaaagcc
     1741 atcgaagcag ctccagccta cacaatcgct caagacgtgt aatgctttta ttatatatta
     1801 gtcacgatat ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg
     1861 aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg
     1921 cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca
     1981 agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat
     2041 tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga
     2101 ctatctttct agggttaaaa aagatttgcg ctttactcga cctaaacttt aaacacgtca
     2161 tagaatcttc gtttgacaaa aaccacattg tggggtaccg agctcgaatt catcgatgat
     2221 atcagatctg ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct
     2281 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     2341 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2401 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2461 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2521 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2581 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2641 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2701 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2761 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2821 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2881 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2941 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3001 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3061 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3121 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3181 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3241 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3301 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3361 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3421 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3481 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3541 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3601 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3661 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3721 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3781 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3841 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3901 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3961 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4021 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4081 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcat
//
