LOCUS       sequence-256818-        6675 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Dox-induced expression of NeuroD1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6675)
  TITLE     Lian Lab CRISPR plasmids
REFERENCE   2  (bases 1 to 6675)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pYJ10-XLone-ND1
FEATURES             Location/Qualifiers
     source          1..6675
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     polyA_signal    1637..1771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2047..2081
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2092..2109)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2092..2108)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2101..2123)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2317..2336)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2436..2458
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2496..2514)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2582..2686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2687..2755,2756..3547)
                     /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(2905..2924)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3718..4306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4207..4226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4460..4477
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4594..4615
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4630..4635,4636..4653,4654..4660)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4668..4684
                     /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)."
     primer_bind     4673..4695
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4692..4708
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4692..4708
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4734..4796
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4972..5053
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5170..5568)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(5569..5625)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(5635..6378)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(6379..6402)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6402..6411
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6431..6642)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta ccatgaccaa atcatacagc gagagcgggc tgatgggcga
      421 gcctcagccc caaggtcccc caagctggac agatgagtgt ctcagttctc aggacgagga
      481 acacgaggca gacaagaaag aggacgagct tgaagccatg aatgcagagg aggactctct
      541 gagaaacggg ggagaggagg aggaggaaga tgaggatcta gaggaagagg aggaagaaga
      601 agaggaggag gaggatcaaa agcccaagag acggggtccc aaaaagaaaa agatgaccaa
      661 ggcgcgccta gaacgtttta aattaaggcg catgaaggcc aacgcccgcg agcggaaccg
      721 catgcacggg ctgaacgcgg cgctggacaa cctgcgcaag gtggtacctt gctactccaa
      781 gacccagaaa ctgtctaaaa tagagacact gcgcttggcc aagaactaca tctgggctct
      841 gtcagagatc ctgcgctcag gcaaaagccc tgatctggtc tccttcgtac agacgctctg
      901 caaaggtttg tcccagccca ctaccaattt ggtcgccggc tgcctgcagc tcaaccctcg
      961 gactttcttg cctgagcaga acccggacat gcccccgcat ctgccaaccg ccagcgcttc
     1021 cttcccggtg catccctact cctaccagtc ccctggactg cccagcccgc cctacggcac
     1081 catggacagc tcccacgtct tccacgtcaa gccgccgcca cacgcctaca gcgcagctct
     1141 ggagcccttc tttgagagcc ccctaactga ctgcaccagc ccttcctttg acggacccct
     1201 cagcccgccg ctcagcatca atggcaactt ctctttcaaa cacgaaccat ccgccgagtt
     1261 tgaaaaaaat tatgccttta ccatgcacta ccctgcagcg acgctggcag ggccccaaag
     1321 ccacggatca atcttctctt ccggtgccgc tgcccctcgc tgcgagatcc ccatagacaa
     1381 cattatgtct ttcgatagcc attcgcatca tgagcgagtc atgagtgccc agcttaatgc
     1441 catctttcac gattagacta gtagaccacc tcccctgcga gctaagctgg acagccaatg
     1501 acgggtaaga gagtgacatt tttcactaac ctaagacagg agggccgtca gagctactgc
     1561 ctaatccaaa gacgggtaaa agtgataaaa atgtatcact ccaacctaag acaggcgcag
     1621 cttccgaggg atttgagatc cagacatgat aagatacatt gatgagtttg gacaaaccaa
     1681 aactagaatg cagtgaaaaa aatgccttat ttgtgaaatt tgtgatgcta ttgccttatt
     1741 tgtaaccatt ataagctgca ataaacaagt ttgatatcta taacaagaaa atatatatat
     1801 aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc
     1861 ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca
     1921 aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg
     1981 agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca
     2041 agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaagaatt cactggccgt
     2101 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     2161 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     2221 acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct
     2281 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
     2341 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     2401 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     2461 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     2521 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     2581 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2641 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2701 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2761 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     2821 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     2881 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     2941 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3001 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3061 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3121 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3181 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3241 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3301 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3361 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3421 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3481 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3541 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3601 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3661 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3721 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3781 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3841 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     3901 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3961 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4021 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4081 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4141 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4201 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4261 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4321 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4381 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4441 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     4501 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4561 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     4621 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     4681 acaatttcac acaggaaaca gctatgacca tgattacgcc aaggtcgact taaccctaga
     4741 aagataatca tattgtgacg tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt
     4801 ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag ttttattata
     4861 tttacactta catactaata ataaattcaa caaacaattt atttatgttt atttatttat
     4921 taaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt agcagtgaaa
     4981 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     5041 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     5101 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatcc
     5161 tctggagatt tagccctccc acacataacc agagggcagc aattcacgaa tcccaactgc
     5221 cgtcggctgt ccatcactgt ccttcactat ggctttgatc ccaggatgca gatcgagaag
     5281 cacctgtcgg caccgtccgc aggggctcaa gatgcccctg ttctcatttc cgatcgcgac
     5341 gatacaagtc aggttgccag ctgccgcagc agcagcagtg cccagcacca cgagttctgc
     5401 acaaggtccc ccagtaaaat gatatacatt gacaccagtg aagatgcggc cgtcgctaga
     5461 gagagctgcg ctggcgacgc tgtagtcttc agagatgggg atgctgttga ttgtagccgt
     5521 tgctctttca atgagggtgg attcttcttg agacaaaggc ttggccatgg ggccggggtt
     5581 ctcctccacg tcgccggcct gcttcagcag gctgaagttg gtggcgccgc tgcccccggg
     5641 gagcatgtca aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa ggtcaaagtc
     5701 gtcaagggca tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt cggtcggccc
     5761 gccgctttcg cactttagct gtttctccag gccacatatg attagttcca ggccgaaaag
     5821 gaaggcaggt tcggctccct gccggtcgaa cagctcaatt gcttgtttca gaagtggggg
     5881 catagaatcg gtggtaggtg tctctctttc ctcttttgct acttgatgct cctgttcctc
     5941 caatacgcag cccagtgtaa agtggcccac ggcggacaga gcgtacagtg cgttctccag
     6001 ggagaagcct tgctgacaca ggaacgcgag ctgattttcc agggtttcgt actgtttctc
     6061 tgttgggcgg gtgccgagat gcactttagc cccgtcgcga tgtgagagga gagcacagcg
     6121 gtatgacttg gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca gggggcagga
     6181 gtgggtatga tgcctgtcca gcatctcgat tggcagggca tcgagcaggg cccgcttgtt
     6241 cttcacgtgc cagtacaggg taggctgctc aactcccagc ttttgagcga gtttccttgt
     6301 cgtcaggcct tcgataccga ctccattgag taattccaga gcagagttta tgactttgct
     6361 cttgtccagt ctagacatct tatcgtcatc gtctttgtaa tccatggtgg cggatcccgc
     6421 gtcacgacac ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta
     6481 ctgcacttat atacggttct cccccaccct cgggaaaaag gcggagccag tacacgacat
     6541 cactttccca gtttaccccg cgccaccttc tctaggcacc ggttcaattg ccgacccctc
     6601 cccccaactt ctcggggact gtgggcgatg tgcgctctgc ccactgacgg gcaccggagc
     6661 cactcgagtg gaatt
//
