LOCUS       sequence-177046-        6181 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express mCherry with a peptide linker (contaning a HCVp 4A/4B 
            substrate site) and a C-terminal CAAX box from KRras for 
            plasma-membrane targeting in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6181)
  AUTHORS   Zhang W, Lohman AW, Zhuravlova Y, Lu X, Wiens MD, Hoi H, Yaganoglu 
            S, Mohr MA, Kitova EN, Klassen JS, Pantazis P, Thompson RJ, Campbell
            RE
  TITLE     Optogenetic control with a photocleavable protein, PhoCl.
  JOURNAL   Nat Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222.
  PUBMED    28288123
REFERENCE   2  (bases 1 to 6181)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222."
COMMENT     Sequence Label: pcDNA-mCherry-4A/4B-CAAX
FEATURES             Location/Qualifiers
     source          1..6181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        124..503
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        504..707
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     658..678
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     752..771
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        752..770
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      795..804
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             813..1520
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(958..976)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1226..1245
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1661..1678)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1667..1891
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1937..2365
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2024..2043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2234..2255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2374..2394)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2379..2708
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2559..2694
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2621..2640
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2775..3569
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2829..2848)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3439..3458
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3743..3864
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3780..3799)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3834..3853
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3913..3929)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3913..3929)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3926..3948)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3937..3953
                     /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(join(3961..3967,3968..3985,3986..3991))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4006..4027
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4144..4161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4315..4903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4395..4414)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5074..5865,5866..5934))
                     /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     5697..5716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5935..6039)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6114..6133)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
       61 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
      121 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      181 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      241 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      301 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      361 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      421 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      481 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      541 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      601 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      661 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
      721 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
      781 ctggctagcg tgccgccacc atggcctcga gaatggtgag caagggcgag gaggataaca
      841 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
      901 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
      961 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     1021 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     1081 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     1141 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     1201 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     1261 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     1321 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     1381 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     1441 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     1501 gcatggacga gctgtacaag ggcagcggca gcgacgagat ggaggagtgc gccagccacc
     1561 tgggcagcgg cagcaaaatg agtaaggatg gaaagaaaaa gaaaaagaaa agcaaaacaa
     1621 agtgtgtaat aatgtaaaag cttggtaaac cgctgatcag cctcgactgt gccttctagt
     1681 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1741 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1801 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     1861 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     1921 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1981 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2041 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2101 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2161 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2221 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2281 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     2341 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     2401 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2461 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2521 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2581 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     2641 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     2701 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     2761 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     2821 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     2881 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     2941 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3001 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3061 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3121 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3181 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3241 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3301 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3361 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3421 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3481 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3541 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     3601 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     3661 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     3721 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     3781 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     3841 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     3901 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3961 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4021 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4081 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4141 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4201 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4261 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4321 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4381 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4441 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4501 gctttctcat agctcacgct ataggtatct cagttcggtg taggtcgttc gctccaagct
     4561 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4621 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4681 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4741 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4801 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4861 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4921 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4981 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5041 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5101 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5161 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5221 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5281 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5341 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5401 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5461 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5521 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5581 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5641 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5701 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5761 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5821 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5881 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5941 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6001 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6061 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     6121 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     6181 g
//
