LOCUS       Exported                7244 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses smURFP and eGFP in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3-smURFP-IRES-eGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7244)
  AUTHORS   Rodriguez EA, Tran GN, Gross LA, Crisp JL, Shu X, Lin JY, Tsien RY
  TITLE     A far-red fluorescent protein evolved from a cyanobacterial 
            phycobiliprotein.
  JOURNAL   Nat Methods. 2016 Aug 1. doi: 10.1038/nmeth.3935.
  PUBMED    27479328
REFERENCE   2  (bases 1 to 7244)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7244
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      917..926
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             923..1324
                     /codon_start=1
                     /product="dimeric small ultra-red fluorescent protein 
                     derived from a cyanobacterial phycobiliprotein (Rodriguez 
                     et al., 2016)"
                     /label=smURFP
                     /translation="MAKTSEQRVNIATLLTENKKKIVDKASQDLWRRHPDLIAPGGIAF
                     SQRDRALCLRDYGWFLHLITFCLLAGDKGPIESIGLISIREMYNSLGVPVPAMMESIRC
                     LKEASLSLLDEEDANETAPYFDYIIKAMS"
     CDS             1918..2637
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1963..1984)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2224..2243)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2571..2592
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2669..2686)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2675..2899
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2945..3373
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3032..3051)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3242..3263
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3382..3402)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3387..3716
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3567..3702
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3629..3648
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3765..4790
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4920..5041
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4957..4976)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5011..5030
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5090..5106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5090..5106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5103..5125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5114..5130
                     /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(5138..5168)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5183..5204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5321..5338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5492..6077)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5572..5591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6248..7108)
                     /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     6871..6890
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7109..7213)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatggctaa gacttccgaa cagagggtga acattgctac
      961 actgctgaca gaaaataaga agaaaatcgt ggataaggct tcccaggatc tgtggcggag
     1021 acacccagac ctgatcgcac caggaggaat tgctttctct cagagggacc gcgctctgtg
     1081 cctgcgagat tacggctggt tcctgcatct gatcaccttt tgtctgctgg ccggagataa
     1141 gggccccatc gagtctattg ggctgatcag tattcgagaa atgtataact cactgggagt
     1201 gcccgtccct gcaatgatgg agagcattag atgcctgaaa gaagccagcc tgtccctgct
     1261 ggacgaagag gacgccaacg agaccgcacc ctactttgat tacattatta aggctatgag
     1321 ctaactcgag tactccggta ttgcggtacc cttgtacgcc tgttttatac tcccttcccg
     1381 taacttagac gcacaaaacc aagttcaata gaagggggta caaaccagta ccaccacgaa
     1441 caagcacttc tgtttccccg gtgatgtcgt atagactgct tgcgtggttg aaagcgacgg
     1501 atccgttatc cgcttatgta cttcgagaag cccagtacca cctcggaatc ttcgatgcgt
     1561 tgcgctcagc actcaacccc agagtgtagc ttaggctgat gagtctggac atccctcacc
     1621 ggtgacggtg gtccaggctg cgttggcggc ctacctatgg ctaacgccat gggacgctag
     1681 ttgtgaacaa ggtgtgaaga gcctattgag ctacataaga atcctccggc ccctgaatgc
     1741 ggctaatccc aacctcggag caggtggtca caaaccagtg attggcctgt cgtaacgcgc
     1801 aagtccgtgg cggaaccgac tactttgggt gtccgtgttt ccttttattt tattgtggct
     1861 gcttatggtg acaatcacag attgttatca taaagcgaat tggataggat ccgtacgatg
     1921 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1981 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2041 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2101 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     2161 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2221 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2281 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2341 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2401 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2461 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2521 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2581 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatct
     2641 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     2701 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     2761 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     2821 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     2881 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     2941 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3001 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3061 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3121 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3181 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3241 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3301 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3361 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     3421 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     3481 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3541 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3601 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     3661 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3721 tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgatgaaa aagcctgaac
     3781 tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga
     3841 tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat
     3901 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
     3961 actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga
     4021 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa
     4081 ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg
     4141 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
     4201 catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
     4261 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg
     4321 aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga
     4381 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc
     4441 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
     4501 cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata
     4561 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg
     4621 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc
     4681 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
     4741 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtgctac
     4801 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4861 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4921 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4981 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5041 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5101 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5161 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5221 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5281 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5641 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6001 ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg caagcagcag attacgcgca
     6061 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6121 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6181 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6241 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6301 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6361 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6421 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6481 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6541 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6601 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6661 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6721 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6781 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6841 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6901 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6961 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7021 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7081 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7141 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7201 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
