LOCUS       sequence-180130-        4403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of shRNA targeting CHRNB4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4403)
  AUTHORS   Oloris SC, Frazer-Abel AA, Jubala CM, Fosmire SP, Helm KM, Robinson 
            SR, Korpela DM, Duckett MM, Baksh S, Modiano JF
  TITLE     Nicotine-mediated signals modulate cell death and survival of T 
            lymphocytes.
  JOURNAL   Toxicol Appl Pharmacol. 2010 Feb 1;242(3):299-309. doi: 
            10.1016/j.taap.2009.10.020. Epub 2009 Nov 4.
  PUBMED    19896492
REFERENCE   2  (bases 1 to 4403)
  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"; doi: 
            "10.1016/j.taap.2009.10.020"; journalName: "Toxicol Appl Pharmacol";
            date: "2010-02-1- 1"; volume: "242"; issue: "3"; pages: "299-309"
COMMENT     Sequence Label: beta4-nAChR shRNA (Beta-4.3)
FEATURES             Location/Qualifiers
     source          1..4403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..215
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     136..155
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(284..300)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(285..301)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(329..347)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(368..384)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(368..384)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(381..403)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    392..408
                     /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(416..422,423..440,441..446))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    461..482
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(599..616)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(770..1358)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(850..869)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1529..2320,2321..2389))
                     /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     2152..2171
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2390..2494)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2520..2975)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2657..2678)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2869..2888
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3102..3124
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3116..3133
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3117..3133
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3140..3159
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3140..3158
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     3191..3207
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3191..3207
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             complement(3270..3869)
                     /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(3353..3373)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3850..3869
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3894..4393)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3942..3961)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     4335..4358
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa
       61 cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc
      121 tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg
      181 gatttgggaa tcttataagt tctgtatgag accacagatc cccttggctg aaacaggatt
      241 caagagattc ctgtttcagc cagacatttt tggaaaagct tatcgatacc gtcgacctcg
      301 agggggggcc cggtaccagc ttttgttccc tttagtgagg gttaatttcg agcttggcgt
      361 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
      421 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
      481 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
      541 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
      601 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
      661 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
      721 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
      781 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
      841 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
      901 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
      961 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1021 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1081 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1141 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1201 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1261 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1321 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1381 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1441 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1501 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1561 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     1621 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     1681 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     1741 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     1801 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     1861 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     1921 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     1981 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2041 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2101 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     2161 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2221 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2281 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2341 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2401 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     2461 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     2521 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     2581 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     2641 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     2701 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     2761 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     2821 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     2881 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     2941 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     3001 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     3061 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     3121 aacgacggcc agtgaattgt aatacgactc actatagggc gaattggagc tccaccgcgg
     3181 tggcggccgc tctagaacta gtggatcccc cgggctgcat ggggtcgtgc gctcctttcg
     3241 gtcgggcgct gcgggtcgtg gggcgggcgt caggcaccgg gcttgcgggt catgcaccag
     3301 gtgcgcggtc cttcgggcac ctcgacgtcg gcggtgacgg tgaagccgag ccgctcgtag
     3361 aaggggaggt tgcggggcgc ggaggtctcc aggaaggcgg gcaccccggc gcgctcggcc
     3421 gcctccactc cggggagcac gacggcgctg cccagaccct tgccctggtg gtcgggcgag
     3481 acgccgacgg tggccaggaa ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct
     3541 tccatctgtt gctgcgcggc cagccgggaa ccgctcaact cggccatgcg cgggccgatc
     3601 tcggcgaaca ccgcccccgc ttcgacgctc tccggcgtgg tccagaccgc caccgcggcg
     3661 ccgtcgtccg cgacccacac cttgccgatg tcgagcccga cgcgcgtgag gaagagttct
     3721 tgcagctcgg tgacccgctc gatgtggcgg tccggatcga cggtgtggcg cgtggcgggg
     3781 tagtcggcga acgcggcggc gagggtgcgt acggccctgg ggacgtcgtc gcgggtggcg
     3841 aggcgcaccg tgggcttgta ctcggtcatg gtaagctagc ttgggctgca ggtcgaaagg
     3901 cccggagatg aggaagagga gaacagcgcg gcagacgtgc gcttttgaag cgtgcagaat
     3961 gccgggcctc cggaggacct tcgggcgccc gccccgcccc tgagcccgcc cctgagcccg
     4021 cccccggacc caccccttcc cagcctctga gcccagaaag cgaaggagca aagctgctat
     4081 tggccgctgc cccaaaggcc tacccgcttc cattgctcag cggtgctgtc catctgcacg
     4141 agactagtga gacgtgctac ttccatttgt cacgtcctgc acgacgcgag ctgcggggcg
     4201 ggggggaact tcctgactag gggaggagta gaaggtggcg cgaaggggcc accaaagaac
     4261 ggagccggtt ggcgcctacc ggtggatgtg gaatgtgtgc gaggccagag gccacttgtg
     4321 tagcgccaag tgcccagcgg ggctgctaaa gcgcatgctc cagactgcct tgggaaaagc
     4381 gcctccccta cccggtagaa ttc
//
