LOCUS       sequence-261419-        7421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Two Color Control Assay bicistronic BFP-mCherry-sTRSVctl.
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: pCS4083
FEATURES             Location/Qualifiers
     source          1..7421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        45..256
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             269..537
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             567..1268
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1325..1344)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1369..1416
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        1645..1749
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1750..1818,1819..2610)
                     /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     complement(1968..1987)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2781..3369
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3270..3289
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3523..3540
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3657..3678
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3693..3698,3699..3716,3717..3723)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3731..3747
                     /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     3736..3758
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3755..3771
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3755..3771
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3820..3941
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3831..3850)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3885..3904
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4071..5093)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(4071..5093)
                     /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
                     "
     protein_bind    complement(5101..5148)
                     /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)."
     primer_bind     5280..5299
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5432..5656
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5645..5662
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5819..6529)
                     /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(6097..6116)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      6526..6535
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6565..6584)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6566..6584)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6629..6832)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6658..6678)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6833..7212
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     7384..7403
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggatcgggag atctggatct gcgatcgctc cggtgcccgt cagtgggcag agcgcacatc
       61 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
      121 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
      181 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
      241 tgccgccaga acacagctga agcttcgagg ggctcgcatc tctccttcac gcgcccgccg
      301 ccctacctga ggccgccatc cacgccggtt gagtcgcgtt ctgccgcctc ccgcctgtgg
      361 tgcctcctga actgcgtccg ccgtctaggt aagtttaaag ctcaggtcga gaccgggcct
      421 ctgtccggcg ctcccttgga gcctacctag actcagccgg ctctccacgc tttgcctgac
      481 cctgcttgct caactctacg tctttgtttc gttttctgtt ctgcgccgtt acagatccaa
      541 gctgtgaccc tcgagtattc gccaccatga gcgagctgat taaggagaac atgcacatga
      601 agctgtacat ggagggcacc gtggacaacc atcacttcaa gtgcacatcc gagggcgaag
      661 gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc ggccctctcc
      721 ccttcgcctt cgacatcctg gctactagct tcctctacgg cagcaagacc ttcatcaacc
      781 acacccaggg catccccgac ttcttcaagc agtccttccc tgagggcttc acatgggaga
      841 gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc agcctccagg
      901 acggctgcct catctacaac gtcaagatca gaggggtgaa cttcacatcc aacggccctg
      961 tgatgcagaa gaaaacactc ggctgggagg ccttcaccga gacgctgtac cccgctgacg
     1021 gcggcctgga aggcagaaac gacatggccc tgaagctcgt gggcgggagc catctgatcg
     1081 caaacatcaa gaccacatat agatccaaga aacccgctaa gaacctcaag atgcctggcg
     1141 tctactatgt ggactacaga ctggaaagaa tcaaggaggc caacaacgag acctacgtcg
     1201 agcagcacga ggtggcagtg gccagatact gcgacctccc tagcaaactg gggcacaagc
     1261 ttaattgatc tgcatgagac gatctcatgc tggagttctt cgcccaccct agggggaggc
     1321 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     1381 gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg
     1441 gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt
     1501 ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg
     1561 gggcggcagg ccctgccata gcctcaggtt actcatagat ctcccgatcc gtcgacgtca
     1621 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1681 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1741 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1801 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1861 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1921 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1981 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2041 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2101 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2161 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2221 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2281 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2341 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2401 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2461 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2521 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2581 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2641 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2701 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2761 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2821 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2881 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     2941 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3001 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3061 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3121 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3181 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3241 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3301 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3361 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3421 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3481 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3541 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     3601 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     3661 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     3721 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     3781 gccaagctct agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg
     3841 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3901 ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc
     3961 agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag
     4021 cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtagtacgtg ctattccttt
     4081 gccctcggac gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca
     4141 tcggtccaga cggccgcgct tctgcgggcg atttgtgtac gcccgacagt cccggctccg
     4201 gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa attgccgtca
     4261 accaagctct gatagagttg gtcaagacca atgcggagca tatacgcccg gagccgcggc
     4321 gatcctgcaa gctccggatg cctccgctcg aagtagcgcg tctgctgctc catacaagcc
     4381 aaccacggcc tccagaagaa gatgttggcg acctcgtatt gggaatcccc gaacatcgcc
     4441 tcgctccagt caatgaccgc tgttatgcgg ccattgtccg tcaggacatt gttggagccg
     4501 aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct cggcccaaag catcagctca
     4561 tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac
     4621 acatggggat cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct
     4681 tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatcgcatcc
     4741 atggcctccg cgaccggctg cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac
     4801 gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg
     4861 tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct
     4921 ttgtagaaac catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg
     4981 aagctgaaag cacgagattc ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg
     5041 aacttttcga tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttggccaag
     5101 gaagttccta tactttctag agaataggaa cttcggaata ggaacttcta ggtacgtgaa
     5161 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     5221 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     5281 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     5341 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtgggg atacccccta
     5401 gagccccagc tggttctttc cgcctcagaa gccatagagc ccaccgcatc cccagcatgc
     5461 ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata
     5521 gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg
     5581 ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg
     5641 caactagaag gcacagtcga ggctgatcag cgggtttggt accaagatta attaaaaaaa
     5701 aaatttttat ttttcttttt gctgttctgt cctcacggac ctcacgtacc ggaaagcaca
     5761 tccggtgaca gctttgtttg tttgcgatcg cggatccggg ccctatattt atgatcagtt
     5821 acttgtacag ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt tcgtactgtt
     5881 ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg acgttgtagg
     5941 ctccgggcag ctgcacgggc ttcttggcct tgtaggtggt cttgacttca gcgtcgtagt
     6001 ggccgccgtc cttcagcttc agcctctgct tgatctcgcc cttcagggcg ccgtcctcgg
     6061 ggtacatccg ctcggaggag gcctcccagc ccatggtctt cttctgcatt acggggccgt
     6121 cggaggggaa gttggtgccg cgcagcttca ctttgtagat gaactcgccg tcctgcaggg
     6181 aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg cgctcccact
     6241 tgaagccctc ggggaaggac agcttcaagt agtcggggat gtcggcgggg tgcttcacgt
     6301 aggccttgga gccgtacatg aactgagggg acaggatgtc ccaggcgaag ggcagggggc
     6361 cacccttggt cactttcagc ttggcggtct gggtgccctc gtaggggcgg ccctcgccct
     6421 cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcact ttgaagcgca
     6481 tgaactcctt gatgatggcc atgttatcct cctcgccctt gctcaccatg gtggcgaccg
     6541 tagcgtgcta gccagcttgg gtctccctat agtgagtcgt attaatttcg ataagccagt
     6601 aagcagtggg ttctctagtt agccagagag ctctgcttat atagacctcc caccgtacac
     6661 gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt
     6721 gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc
     6781 gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa
     6841 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact
     6901 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
     6961 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
     7021 gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
     7081 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
     7141 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca
     7201 ataatcaatg tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa
     7261 ccctaagcag attcttcatg caattgtcgg tcaagccttg ccttgttgta gcttaaattt
     7321 tgctcgcgca ctactcagcg acctccaaca cacaagcagg gagcagatac tggcttaact
     7381 atgcggcatc agagcagatt gtactgagag tgcaccatag g
//
