LOCUS       Exported               10656 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pLB2 CAG P2Gm
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10656)
  AUTHORS   Stern P, Astrof S, Erkeland SJ, Schustak J, Sharp PA, Hynes RO
  TITLE     A system for Cre-regulated RNA interference in vivo.
  JOURNAL   Proc Natl Acad Sci U S A. 2008 Sep 16. 105(37):13895-900.
  PUBMED    18779577
REFERENCE   2  (bases 1 to 10656)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2008 Sep 16. 105(37):13895-900."
FEATURES             Location/Qualifiers
     source          1..10656
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     355..372
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    489..510
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        525..555
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    563..579
                     /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     568..590
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     587..603
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     587..603
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     LTR             869..1049
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    1096..1221
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1720..1953
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2138..2182
                     /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    2480..2597
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        3119..3422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3424..3701
                     /label=chicken beta-actin promoter
     intron          3703..4720
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4644..4666
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4728..4747
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      4809..4818
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4815..5411
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MVEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     5311..5331
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5433..5498
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     regulatory      5508..5517
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             5514..6230
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5559..5580)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5820..5839)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6167..6188
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           6283..6404
                     /label=5 miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           6431..6445
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           6473..6599
                     /label=3 miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     misc_feature    6685..7273
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6738..6758)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7156..7167)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7276..7292)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7277..7293)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    8436..8643
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     LTR             8662..8842
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(8904..9492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8984..9003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(9663..10523)
                     /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     10286..10305
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10524..10628)
                     /gene="bla"
                     /label=AmpR promoter