LOCUS       Exported                9944 bp ds-DNA     circular SYN 06-APR-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pWHERE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9944)
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9944
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    complement(1..2032)
                     /note="H19 ins"
                     /note="mH19 insulators on either side of the lacZ 
                     transcription unit. Both insulators are expected to protect
                     the integrated transcriptional LacZ unit from negative as 
                     well as positive influences from neighboring sequences. 
                     Insulator elements can be functionally identified by their 
                     ability to shield promoters from regulators in a 
                     position-dependent manner or by their ability to protect 
                     adjacent transgenes from position effects. The fragment of 
                     the differential methylated region (DMD) located between 
                     the mouse Igf2 and H19 acts as a powerful insulator. The 
                     enhancer blocking activity of the DMD fragment is dependent
                     upon four responsive elements to the vertebrate 
                     enhancer-blocking protein CTCF. Two mouse DMD fragments 
                     have been introduced in opposite orientation in the pWHERE 
                     plasmid to insulate your promoter of interest cloned 
                     upstream of the new CpG-free LacZ gene from the 5 and 3 
                     adjacent regions at the integrated site in transgenic 
                     mice."
     CDS             2071..5157
                     /codon_start=1
                     /product="The E. coli lacZ gene codes for the enzyme 
                     beta-galactosidase which catalyzes the hydrolysis of the 
                     substrate X-Gal to produce a blue color that is easily 
                     visualized under a microscope. A nuclear localization 
                     signal of SV40 large T has been inserted in the 5 end of 
                     the lacZ gene to allow the targeting of the chimeric 
                     protein to the nucleus. To reduce the immunogenicity of 
                     this bacterial gene, InvivoGen has engineered a synthetic 
                     lacZnls gene that is entirely free of CpG motifs, whereas 
                     the wild type lacZ gene contains 298 CpG dinucleotides."
                     /note="LacZ Delta-CpG NLS"
                     /translation="MDPVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQ
                     LRSLNGEWRFAWFPAPEAVPESWLECDLPEAVPKKKRKVEADTVVVPSNWQMHGYDAPI
                     YTNVTYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRW
                     VGYGQDSRLPSEFDLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHK
                     PTTQISDFHVATRFNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGG
                     EIIDERGGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFREV
                     RIENGLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSHYPN
                     HPLWYTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQRDRNHPSV
                     IIWSLGNESGHGANHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPMYARVDEDQPF
                     PAVPKWSIKKWLSLPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQYPRLQGGFVWDWV
                     DQSLIKYDENGNPWSAYGGDFGDTPNDRQFCMNGLVFADRTPHPALTEAKHQQQFFQFR
                     LSGQTIEVTSEYLFRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIELPELPQPE
                     SAGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPHLTTSEMDFC
                     IELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIGVSEATRIDPNAWV
                     ERWKAAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGKTLFISRKTYRIDGSGQMAI
                     TVDVEVASDTPHPARIGLNCQLAQVAERVNWLGLGPQENYPDRLTAACFDRWDLPLSDM
                     YTPYVFPSENGLRCGTRELNYGPHQWRGDFQFNISRYSQQQLMETSHRHLLHAEEGTWL
                     NIDGFHMGIGGDDSWSPSVSAEFQLSAGRYHYQLVWCQK"
     CDS             2302..2322
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     polyA_signal    5170..5742
                     /note="EF-1-alpha poly(A) signal"
                     /note="human EF-1-alpha polyadenylation signal"
     misc_feature    complement(5750..7781)
                     /note="H19 ins"
                     /note="mH19 insulators on either side of the lacZ 
                     transcription unit. Both insulators are expected to protect
                     the integrated transcriptional LacZ unit from negative as 
                     well as positive influences from neighboring sequences. 
                     Insulator elements can be functionally identified by their 
                     ability to shield promoters from regulators in a 
                     position-dependent manner or by their ability to protect 
                     adjacent transgenes from position effects. The fragment of 
                     the differential methylated region (DMD) located between 
                     the mouse Igf2 and H19 acts as a powerful insulator. The 
                     enhancer blocking activity of the DMD fragment is dependent
                     upon four responsive elements to the vertebrate 
                     enhancer-blocking protein CTCF. Two mouse DMD fragments 
                     have been introduced in opposite orientation in the pWHERE 
                     plasmid to insulate your promoter of interest cloned 
                     upstream of the new CpG-free LacZ gene from the 5 and 3 
                     adjacent regions at the integrated site in transgenic 
                     mice."
     rep_origin      complement(7877..8465)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8636..9496)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(9497..9601)
                     /gene="bla"
                     /note="AmpR promoter"