LOCUS       Exported                9886 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  3rd generation lentiviral vector, expresses Ngn2 and puromycin 
            resistent gene under control of TetON promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pTet-O-Ngn2-puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9886)
  AUTHORS   Zhang Y, Pak C, Han Y, Ahlenius H, Zhang Z, Chanda S, Marro S, 
            Patzke C, Acuna C, Covy J, Xu W, Yang N, Danko T, Chen L, Wernig M, 
            Sudhof TC
  TITLE     Rapid single-step induction of functional neurons from human 
            pluripotent stem cells.
  JOURNAL   Neuron. 2013 Jun 5;78(5):785-98. doi: 10.1016/j.neuron.2013.05.029.
  PUBMED    23764284
REFERENCE   2  (bases 1 to 9886)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.neuron.2013.05"; journalName: "Neuron"; date: "2013-06-5-
            5"; volume: "78"; issue: "5"; pages: "785-98"
FEATURES             Location/Qualifiers
     source          1..9886
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        100..479
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        481..679
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     634..654
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             697..877
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    924..1049
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1542..1775
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1960..2004
                     /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    2302..2419
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2476..2746
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        2779..2817
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2814..2838
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2917..2926
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2926..2949
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3742..3795
                     /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             3802..4401
                     /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(3802..3821)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4298..4318
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4440..5028
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4493..4513)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4911..4922)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5031..5047)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5032..5048)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5553..5733
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5759..5776)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5765..5989
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6035..6463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6122..6141)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6332..6353
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6472..6492)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6477..6806
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6657..6792
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6719..6738
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6854..6901
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6920..7294
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    7424..7545
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7461..7480)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7515..7534
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7594..7610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7594..7610)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7607..7629)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7618..7634
                     /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(7642..7672)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7687..7708
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7825..7842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7996..8584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8076..8095)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(8755..9615)
                     /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     9378..9397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9616..9720)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9795..9814)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"