LOCUS       sequence-466097-        6230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DOX-inducible U6 driven shRNA against mouse Esr1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6230)
  AUTHORS   Golden CEM, Martin AC, Kaur D, Mah A, Levy DH, Yamaguchi T, Lasek 
            AW, Lin D, Aoki C, Constantinople CM
  TITLE     Estrogenic control of reward prediction errors and reinforcement 
            learning.
  JOURNAL   bioRxiv [Preprint]. 2024 Sep 25:2023.12.09.570945. doi: 
            10.1101/2023.12.09.570945.
  PUBMED    38105956
REFERENCE   2  (bases 1 to 6230)
  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: "bioRxiv 
            [Preprint]. 2024 Sep 25:2023.12.09.570945. doi: 
            10.1101/2023.12.09.570945."
COMMENT     Sequence Label: AAV-U6/TO-shEsr1-CMV-TetR-eGFP
FEATURES             Location/Qualifiers
     source          1..6230
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     146..166
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     317..336
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    347..365
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    375..393
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        463..766
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        767..970
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     921..941
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     967..991
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1118..1741
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1194..1213)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1760..1813
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1814..1816,1817..2527)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1814..1816,1817..2527)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1856..1877)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2117..2136)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2464..2485
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2819..3407
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2872..2892)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3290..3301)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3494..3634
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3494..3623
                     /label=AAV2 ITR
     rep_origin      3709..4164
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3796..3815)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4006..4027
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4181..4200)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4300..4322
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4360..4378)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4446..4550
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4551..4619,4620..5411)
                     /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     complement(4769..4788)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5582..6170
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6071..6090
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt catatttgca
      181 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
      241 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
      301 attatgtttt aaaatggact atcatatgct taccgtaact tgaaactccc tatcagtgat
      361 agagattata tatctcccta tcagtgatag agaccggcat ggagcatctc tacacttcct
      421 gtcatgtaga gatgctccat gcctttttta ccgtctagag tccgttacat aacttacggt
      481 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      541 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      601 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      661 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      721 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      781 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      841 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      901 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      961 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
     1021 cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac
     1081 ggacctgcag gaattgatcc gcggccgcac cggtaccatg atgtccagat tagataaaag
     1141 taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag gtttaacaac
     1201 ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa
     1261 taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc atactcactt
     1321 ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta aaagttttag
     1381 atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac ggcctacaga
     1441 aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag gtttttcact
     1501 agagaatgca ttatatgcac tcagcgctgt ggggcatttt actttaggtt gcgtattgga
     1561 agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg atagtatgcc
     1621 gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc cagccttctt
     1681 attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc
     1741 cgcggctagc ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga
     1801 gaatcctggc ccagtgagca agggcgagga gctgttcacc ggggtggtac ccatcctggt
     1861 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1921 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1981 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2041 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2101 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     2161 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2221 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     2281 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     2341 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2401 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2461 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2521 gtacaagtcc ggactcagat ctcgagagga ggaggaggag acagacagca ggatgcccca
     2581 cctcgacagc cccggcagct cccagccgag acgctccttc ctctcaaggg tgatcagggc
     2641 agcgctaccg ttgcagctgc ttctgctgct gctgctgctc ctggcctgcc tgctacctgc
     2701 ctctgaagat gactacagct gcacccaggc caacaacttt gcccgatcct tctaccccat
     2761 gctgcggtac accaacgggc cacctcccac ctaggaattc gatatcaagc ttatcgataa
     2821 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     2881 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     2941 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3001 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3061 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3121 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3181 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     3241 ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3301 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3361 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc
     3421 gctaataaaa gatctttatt ttcattagat ctgtgtgttg gttttttgtg tcacgtgcgg
     3481 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     3541 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     3601 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     3661 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     3721 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3781 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3841 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3901 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     3961 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4021 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     4081 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4141 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     4201 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4261 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4321 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     4381 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4441 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4501 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4561 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4621 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4681 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4741 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4801 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4861 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4921 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4981 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5041 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5101 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5161 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5221 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5281 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5341 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5401 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5461 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5521 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5581 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5641 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5701 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5761 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5821 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5881 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5941 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6001 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6061 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6121 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6181 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
//
