LOCUS       sequence-157852-        7609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses an shRNA against NKCC1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7609)
  AUTHORS   Young SZ, Taylor MM, Wu S, Ikeda-Matsuo Y, Kubera C, Bordey A
  TITLE     NKCC1 Knockdown Decreases Neuron Production through GABAA-Regulated 
            Neural Progenitor Proliferation and Delays Dendrite Development.
  JOURNAL   J Neurosci. 2012 Sep 26;32(39):13630-8.
  PUBMED    23015452
REFERENCE   2  (bases 1 to 7609)
  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: "J 
            Neurosci."; date: "2012-09-26"; volume: "32(39)"; pages: "13630-8"
COMMENT     Sequence Label: pSico-shNKCC1
FEATURES             Location/Qualifiers
     source          1..7609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1686..1919
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2104..2148
                     /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"
     misc_feature    2446..2563
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     2854..2874
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(2892..2925)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     enhancer        3025..3328
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3329..3532
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      3571..3580
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3577..3579,3580..3582,3583..4296)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3577..3579,3580..3582,3583..4293)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3622..3643)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3883..3902)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4230..4251
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4304..4337)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     primer_bind     4407..4423
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    4498..5086
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4551..4571)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4969..4980)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5089..5105)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             5615..5795
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5857..6445)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5937..5956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6616..7407,7408..7476))
                     /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     7239..7258
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7477..7581)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga
     1561 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1621 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1681 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1741 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1801 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1861 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1921 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1981 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     2041 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     2101 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     2161 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     2221 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     2281 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     2341 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     2401 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2581 cagcagagat ccagtttggt tagtaccggg cccgctctag agatccgacg ccgccatctc
     2641 taggcccgcg ccggccccct cgcacagact tgtgggagaa gctcggctac tcccctgccc
     2701 cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac
     2761 agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa
     2821 gagatacaaa tactaaatta ttattttaaa aaacagcaca aaaggaaact caccctaact
     2881 gtaaagtaat tataacttcg tatagtataa attatacgaa gttataagcc ttggtttttt
     2941 gaattccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt
     3001 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     3061 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     3121 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     3181 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     3241 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     3301 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     3361 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     3421 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     3481 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     3541 gaaccgtcag atccgctagc gctaccggtc gccaccatgg tgagcaaggg cgaggagctg
     3601 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     3661 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     3721 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     3781 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     3841 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     3901 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3961 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     4021 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     4081 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     4141 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     4201 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     4261 gggatcactc tcggcatgga cgagctgtac aagtagcggc cgcataactt cgtatagtat
     4321 aaattatacg aagttataag ccttgtttga cacacttgtc ctgggatttt gatatccgaa
     4381 tcccaggaca agtgtgtctt ttttctcgag gtcgacggta tcgataagct cgcttcacga
     4441 gatcatgttt aagggttccg gttccactag gtacaattcg atatcaagct tatcgataat
     4501 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     4561 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     4621 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     4681 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     4741 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     4801 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     4861 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     4921 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     4981 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     5041 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     5101 cctcgatcga gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa
     5161 tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc
     5221 tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     5281 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct
     5341 gtggatctac cacacacaag gctacttccc tgattggcag aactacacac cagggccagg
     5401 gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa
     5461 ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg
     5521 gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca
     5581 tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag accagatctg
     5641 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     5701 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     5761 cagacccttt tagtcagtgt ggaaaatctc tagcagcatg tgagcaaaag gccagcaaaa
     5821 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5881 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5941 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6001 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6061 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6121 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6181 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6241 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6301 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6361 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6421 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6481 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6541 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6601 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6661 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6721 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6781 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6841 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6901 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6961 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7021 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7081 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7141 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7201 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7261 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7321 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7381 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7441 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7501 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7561 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgac
//
