LOCUS       Exported               10442 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  AAVS1 safe harbor targeting vector with blasticidin resistance gene 
            expressing AtAFB2-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    pSH-EFIRES-B-AtAFB2-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10442)
  AUTHORS   Li S, Prasanna X, Salo VT, Vattulainen I, Ikonen E
  TITLE     An efficient auxin-inducible degron system with low basal 
            degradation in human cells.
  JOURNAL   Nat Methods. 2019 Aug 26. pii: 10.1038/s41592-019-0512-x. doi: 
            10.1038/s41592-019-0512-x.
  PUBMED    31451765
REFERENCE   2  (bases 1 to 10442)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2019 Aug 26. pii: 10.1038/s41592-019-0512-x. doi: 
            10.1038/s41592-019-0512-x."
FEATURES             Location/Qualifiers
     source          1..10442
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(135..152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(306..894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(386..405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1065..1925)
                     /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     1688..1707
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1926..2030)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2057..2512
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2144..2163)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2354..2375
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    3029..3832
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     polyA_signal    3858..3906
                     /note="synthetic polyadenylation signal"
     misc_feature    3920..4011
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     3960..3979
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        4023..5185
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4254..5176
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5133..5153
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     intron          5224..5356
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     5339..5358
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        5401..5419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             7186..7893
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(7328..7346)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7596..7615
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    7928..8480
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(8072..8089)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     8299..8318
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             8480..8881
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MGAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFT
                     GVNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPG
                     IKAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    9039..9160
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9076..9095)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9130..9149
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    9196..10032
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"