LOCUS       sequence-252729-        7421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Two Color Control Assay bicistronic BFP-mCherry-sTRSV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7421)
  AUTHORS   Xiang JS, Kaplan M, Dykstra P, Hinks M, McKeague M, Smolke CD
  TITLE     Massively parallel RNA device engineering in mammalian cells with 
            RNA-Seq.
  JOURNAL   Nat Commun. 2019 Sep 23;10(1):4327. doi: 10.1038/s41467-019-12334-y.
  PUBMED    31548547
REFERENCE   2  (bases 1 to 7421)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2019-09-23"; volume: "10(1):4327. doi"; pages: " 
            10.1038/s41467-019-12334-y"
COMMENT     Sequence Label: pCS4084
FEATURES             Location/Qualifiers
     source          1..7421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        48..259
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             272..540
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             570..1271
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1328..1347)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1372..1419
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(1632..1651)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1823..2202
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2203..2406
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2357..2377
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2451..2470
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2451..2469
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2500..2509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2506..3216
                     /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     2919..2938
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_RNA        3264..3313
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     primer_bind     complement(3373..3390)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3379..3603
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3736..3755)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3887..3934
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3942..4964
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3942..4964
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5094..5215
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5131..5150)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5185..5204
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5264..5280)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5264..5280)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5277..5299)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5288..5304
                     /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(5312..5318,5319..5336,5337..5342))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5357..5378
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5495..5512)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5666..6254)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5746..5765)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6425..7216,7217..7285))
                     /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     7048..7067
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7286..7390)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctgga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac
       61 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
      121 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
      181 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
      241 gtttgccgcc agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg
      301 ccgccctacc tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg
      361 tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg
      421 cctctgtccg gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct
      481 gaccctgctt gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc
      541 caagctgtga ccctcgagta ttcgccacca tgagcgagct gattaaggag aacatgcaca
      601 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
      661 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
      721 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
      781 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
      841 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
      901 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
      961 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     1021 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     1081 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     1141 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg
     1201 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     1261 agcttaattg atctgcatga gacgatctca tgctggagtt cttcgcccac cctaggggga
     1321 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     1381 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     1441 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     1501 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     1561 tcggggcggc aggccctgcc atagcctcag gttactcata gatctcccga tcccctatgg
     1621 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     1681 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     1741 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     1801 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     1861 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     1921 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     1981 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     2041 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     2101 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     2161 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     2221 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     2281 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     2341 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     2401 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
     2461 cactataggg agacccaagc tggctagcac gctacggtcg ccaccatggt gagcaagggc
     2521 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaagtgca catggagggc
     2581 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     2641 acccagaccg ccaagctgaa agtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     2701 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     2761 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     2821 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     2881 aaagtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     2941 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     3001 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaagt caagaccacc
     3061 tacaaggcca agaagcccgt gcagctgccc ggagcctaca acgtcaacat caagttggac
     3121 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     3181 cactccaccg gcggcatgga cgagctgtac aagtaactga tcataaatat agggcccgga
     3241 tccgcgatcg caaacaaaca aagctgtcac cggatgtgct ttccggtctg atgagtccgt
     3301 gaggacgaaa cagcaaaaag aaaaataaaa attttttttt taattaatct tggtaccaaa
     3361 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3421 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3481 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3541 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3601 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3661 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3721 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3781 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3841 acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc
     3901 gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg
     3961 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     4021 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     4081 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     4141 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     4201 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     4261 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     4321 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     4381 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     4441 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     4501 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     4561 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     4621 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     4681 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     4741 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     4801 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     4861 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     4921 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt
     4981 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5041 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5101 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5161 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5221 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5281 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5341 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5401 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5461 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5521 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5581 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5641 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5701 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5761 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5821 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5881 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5941 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6001 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6061 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     6121 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6181 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6241 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6301 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6361 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6421 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6481 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6541 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6601 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6661 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6721 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6781 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6841 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6901 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6961 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7021 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7081 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7141 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7201 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7261 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7321 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7381 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
