LOCUS       Exported                9174 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  lentiviral luciferase reporter containing CDH1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    7TFP CDH1 reporter
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9174)
  AUTHORS   Pattabiraman DR, Bierie B, Kober KI, Thiru P, Krall JA, Zill C, 
            Reinhardt F, Tam WL, Weinberg RA
  TITLE     Activation of PKA leads to mesenchymal-to-epithelial transition and 
            loss of tumor-initiating ability.
  JOURNAL   Science. 2016 Mar 4;351(6277):aad3680. doi: 10.1126/science.aad3680.
  PUBMED    26941323
REFERENCE   2  (bases 1 to 9174)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2016-03-4"; volume: "351(6277):aad3680. doi"; pages: " 
            10.1126/science.aad3680"
FEATURES             Location/Qualifiers
     source          1..9174
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        325..356
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      412..421
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             418..2070
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(484..502)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(2089..2109)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2094..2423
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2274..2409
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2433..3032
                     /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(2433..2452)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2929..2949
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3099..3687
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3152..3172)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3570..3581)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3774..4007
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4079..4200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4116..4135)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4170..4189
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4240..4375
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4395..4414)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4396..4414)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4424..4441)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4424..4440)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4433..4455)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4582..5037
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4669..4688)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4879..4900
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5063..5167
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5168..6028
                     /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(5386..5405)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6199..6787
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6688..6707
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6941..6958
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7075..7096
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7111..7141
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7149..7165
                     /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     7154..7176
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7173..7189
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7173..7189
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7208..7228
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7210..7228
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7256..7482
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7483..7663
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    7710..7835
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8328..8561
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8746..8790
                     /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    9057..9174
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"