LOCUS       sequence-246295-        8012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of mouse Sik3 shRNA with GFP and puromycin 
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8012)
  AUTHORS   Tarumoto Y, Lin S, Wang J, Milazzo JP, Xu Y, Lu B, Yang Z, Wei Y, 
            Polyanskaya S, Wunderlich M, Gray NS, Stegmaier K, Vakoc CR
  TITLE     Salt-Inducible Kinase inhibition suppresses acute myeloid leukemia 
            progression in vivo.
  JOURNAL   Blood. 2019 Nov 1. pii: 422697. doi: 10.1182/blood.2019001576.
  PUBMED    31697837
REFERENCE   2  (bases 1 to 8012)
  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"; journalName: "Blood. 2019
            Nov 1. pii: 422697. doi: 10.1182/blood.2019001576."
COMMENT     Sequence Label: LEPG_shSik3.3974
FEATURES             Location/Qualifiers
     source          1..8012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           1592..1606
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        1790..2289
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1825..1848)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2222..2241
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2310..2909
                     /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(2310..2329)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2806..2826
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2970..3537
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2987..3539
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3131..3148)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3358..3377
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3551..3553,3554..3556,3557..4270)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3596..3617)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3857..3876)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4204..4225
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4372..4886
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5081..5097
                     /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(join(5105..5111,5112..5129,5130..5135))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5150..5171
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5288..5305)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5459..6047)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5539..5558)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6218..7009,7010..7078))
                     /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     6841..6860
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7079..7183)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7251..7269
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7307..7329)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7429..7448
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7642..7664
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7792..7811
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(8011..8012,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctctcga ctagggataa cagggtaatt
     1441 gtttgaatga ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc
     1501 tgggattact tcgacttctt aacccaacag aaggctcgag aaggtatatt gctgttgaca
     1561 gtgagcgcca ggatgaggaa gatgaagaat agtgaagcca cagatgtatt cttcatcttc
     1621 ctcatcctga tgcctactgc ctcggacttc aaggggctag aattcgagca attatcttgt
     1681 ttactaaaac tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat
     1741 ggtataaatt aaatcacttt tttcaattga cgcgtgatct aattctaccg ggtaggggag
     1801 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
     1861 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
     1921 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
     1981 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt
     2041 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
     2101 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg
     2161 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg
     2221 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg
     2281 ggcctttcga cctgcagccc aagcttacca tgaccgagta caagcccacg gtgcgcctcg
     2341 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     2401 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     2461 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     2521 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     2581 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     2641 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     2701 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     2761 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     2821 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     2881 ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg
     2941 aaaggagcgc acgaccccat gcatccaatt ccgccccccc cccctaacgt tactggccga
     3001 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     3061 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     3121 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     3181 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     3241 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     3301 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     3361 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     3421 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
     3481 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat
     3541 ggccacaacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     3601 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga
     3661 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     3721 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     3781 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     3841 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     3901 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3961 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     4021 gcagaagaac ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt
     4081 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     4141 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     4201 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     4261 gtacaagtaa tgaattaatt aagaattatc aagcttatcg ataccgtcga cctgcagcca
     4321 agcttatcga taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac
     4381 cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata
     4441 cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg
     4501 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     4561 tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg
     4621 ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg
     4681 cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg
     4741 gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtgtatccaa taaaccctct
     4801 tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     4861 actacccgtc agcgggggtc tttcatgggt aacagtttct tgaagttgga gaacaacatt
     4921 ctgagggtag gagtcgaata ttaagtaatc ctgactcaat tagccactgt tttgaatcca
     4981 catactccaa tactcctgaa atagttcatt atggacagcg cagaaagagc tggggagaat
     5041 tgtgaaattg tcacagaaag agctggggag aattgtgaaa ttgttatccg ctcacaattc
     5101 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5161 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5221 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5281 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5341 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5401 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5461 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5521 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5581 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5641 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5701 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5761 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5821 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5881 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5941 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6001 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6061 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6121 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6181 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6241 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6301 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6361 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6421 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6481 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6541 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6601 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6661 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6721 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6781 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6841 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6901 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6961 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7021 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7081 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7141 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7201 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     7261 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     7321 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     7381 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
     7441 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     7501 aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     7561 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     7621 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcg caaggaatgg
     7681 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     7741 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     7801 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     7861 ggcgtagagg cgattagtcc aatttgttaa agacaggata tcagtggtcc aggctctagt
     7921 tttgactcaa caatatcacc agctgaagcc tatagagtac gagccataga taaaataaaa
     7981 gattttattt agtctccaga aaaagggggg aa
//
