LOCUS       Exported               12352 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  3rd generation lentiviral vector encoding FUCCI reporters for cell 
            cycle analysis and puro for selection.
ACCESSION   .
VERSION     .
KEYWORDS    pBOB-EF1-FastFUCCI-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12352)
  AUTHORS   Koh SB, Mascalchi P, Rodriguez E, Lin Y, Jodrell DI, Richards FM, 
            Lyons SK
  TITLE     Quantitative FastFUCCI assay defines cell cycle dynamics at 
            single-cell level.
  JOURNAL   J Cell Sci. 2016 Nov 25. pii: jcs.195164.
  PUBMED    27888217
REFERENCE   2  (bases 1 to 12352)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell Sci.
            2016 Nov 25. pii: jcs.195164."
FEATURES             Location/Qualifiers
     source          1..12352
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        172..551
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        553..751
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     706..726
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             769..949
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    996..1121
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1618..1851
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2036..2080
                     /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    2332..2448
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2465..3643
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2696..3634
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3591..3611
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3664..4314
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAH"
     CDS             4369..4641
                     /codon_start=1
                     /product="degron consisting of residues 30-120 of human 
                     Cdt1 (Zielke and Edgar, 2015)"
                     /label=Cdt1 (30-120)
                     /translation="PSPARPALRAPASATSGSRKRARPPAAPGRDQARPPARRRLRLSV
                     DEVSSPSTPEAPDIPACPSPGQKIKKSTPAAGQPPHLTSAQDQDTI"
     CDS             4648..4701
                     /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             4708..5385
                     /codon_start=1
                     /product="monomeric Azami-Green fluorescent protein 
                     (Karasawa et al., 2003)"
                     /label=mAzami-Green
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             5416..5745
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
     promoter        5788..6287
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5823..5846)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6220..6239
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6331..6930
                     /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(6331..6350)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6827..6847
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7092..7680
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7145..7165)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7563..7574)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8091..8271
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(8297..8314)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8303..8527
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8573..9001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8660..8679)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8870..8891
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9010..9030)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9015..9344
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9195..9330
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9257..9276
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9392..9439
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9458..9832
                     /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    9962..10083
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9999..10018)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10053..10072
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10132..10148)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10132..10148)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10145..10167)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10156..10172
                     /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(10180..10210)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10225..10246
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10363..10380)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10534..11122)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10614..10633)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(11293..12153)
                     /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     11916..11935
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12154..12258)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12333..12352)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"