LOCUS       sequence-180128-        4409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of shRNA targeting CHRNA4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4409)
  TITLE     Modiano Lab plasmids
REFERENCE   2  (bases 1 to 4409)
  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: alpha4-nAChR shRNA (Alpha-4.3)
FEATURES             Location/Qualifiers
     source          1..4409
                     /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(290..306)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(291..307)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(335..353)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(374..390)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(374..390)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(387..409)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    398..414
                     /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(422..428,429..446,447..452))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    467..488
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(605..622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(776..1364)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(856..875)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1535..2326,2327..2395))
                     /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     2158..2177
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2396..2500)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2526..2981)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2663..2684)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2875..2894
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3108..3130
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3122..3139
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3123..3139
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3146..3165
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3146..3164
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     3197..3213
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3197..3213
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             complement(3276..3875)
                     /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(3359..3379)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3856..3875
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3900..4399)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3948..3967)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     4341..4364
                     /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 ccatgagaag aaccagatga
      241 tgattaagag atcatcatct ggttcttctc attttttgga aaagcttatc gataccgtcg
      301 acctcgaggg ggggcccggt accagctttt gttcccttta gtgagggtta atttcgagct
      361 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
      421 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
      481 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
      541 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
      601 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
      661 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
      721 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
      781 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
      841 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
      901 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
      961 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1021 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1081 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1141 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1201 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1261 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1321 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     1381 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     1441 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     1501 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     1561 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     1621 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     1681 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     1741 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     1801 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     1861 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     1921 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     1981 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     2041 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     2101 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     2161 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     2221 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     2281 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     2341 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     2401 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     2461 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     2521 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2581 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2641 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2701 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     2761 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     2821 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     2881 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     2941 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
     3001 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     3061 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     3121 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcca
     3181 ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcatgggg tcgtgcgctc
     3241 ctttcggtcg ggcgctgcgg gtcgtggggc gggcgtcagg caccgggctt gcgggtcatg
     3301 caccaggtgc gcggtccttc gggcacctcg acgtcggcgg tgacggtgaa gccgagccgc
     3361 tcgtagaagg ggaggttgcg gggcgcggag gtctccagga aggcgggcac cccggcgcgc
     3421 tcggccgcct ccactccggg gagcacgacg gcgctgccca gacccttgcc ctggtggtcg
     3481 ggcgagacgc cgacggtggc caggaaccac gcgggctcct tgggccggtg cggcgccagg
     3541 aggccttcca tctgttgctg cgcggccagc cgggaaccgc tcaactcggc catgcgcggg
     3601 ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg gcgtggtcca gaccgccacc
     3661 gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga gcccgacgcg cgtgaggaag
     3721 agttcttgca gctcggtgac ccgctcgatg tggcggtccg gatcgacggt gtggcgcgtg
     3781 gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg ccctggggac gtcgtcgcgg
     3841 gtggcgaggc gcaccgtggg cttgtactcg gtcatggtaa gctagcttgg gctgcaggtc
     3901 gaaaggcccg gagatgagga agaggagaac agcgcggcag acgtgcgctt ttgaagcgtg
     3961 cagaatgccg ggcctccgga ggaccttcgg gcgcccgccc cgcccctgag cccgcccctg
     4021 agcccgcccc cggacccacc ccttcccagc ctctgagccc agaaagcgaa ggagcaaagc
     4081 tgctattggc cgctgcccca aaggcctacc cgcttccatt gctcagcggt gctgtccatc
     4141 tgcacgagac tagtgagacg tgctacttcc atttgtcacg tcctgcacga cgcgagctgc
     4201 ggggcggggg ggaacttcct gactagggga ggagtagaag gtggcgcgaa ggggccacca
     4261 aagaacggag ccggttggcg cctaccggtg gatgtggaat gtgtgcgagg ccagaggcca
     4321 cttgtgtagc gccaagtgcc cagcggggct gctaaagcgc atgctccaga ctgccttggg
     4381 aaaagcgcct cccctacccg gtagaattc
//
