LOCUS       sequence-51778-e        8282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8282)
  AUTHORS   Lam AJ, St-Pierre F, Gong Y, Marshall JD, Cranfill PJ, Baird MA, 
            McKeown MR, Wiedenmann J, Davidson MW, Schnitzer MJ, Tsien RY, Lin 
            MZ
  TITLE     Improving FRET dynamic range with bright green and red fluorescent 
            proteins.
  JOURNAL   Nat Methods. 2012 Sep 9. doi: 10.1038/nmeth.2171.
  PUBMED    22961245
REFERENCE   2  (bases 1 to 8282)
  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 
            Methods. 2012 Sep 9. doi: 10.1038/nmeth.2171."
COMMENT     Sequence Label: pcDNA3.1/Puro-CAG-VSFP-CR
FEATURES             Location/Qualifiers
     source          1..8282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        15..394
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        396..669
                     /label=chicken beta-actin promoter
     intron          670..1686
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1610..1632
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1694..1713
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1845..1854
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2658..2679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             3315..4025
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     complement(4143..4160)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4149..4373
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4419..4847
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4506..4525)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4716..4737
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4856..4876)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4861..5190
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5041..5176
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5103..5122
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5199..5798
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5199..5218)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5695..5715
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5958..6079
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5995..6014)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6049..6068
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6128..6144)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6128..6144)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6141..6163)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6152..6168
                     /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(6176..6182,6183..6200,6201..6206))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6221..6242
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6359..6376)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6530..7115)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6610..6629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7286..8077,8078..8146))
                     /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     7909..7928
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8147..8251)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
       61 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      121 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      181 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      241 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      301 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      361 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
      421 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
      481 attttgtgca gcgatggggg cggggggggg gggcgcgcgc caggcggggc ggggcggggc
      541 gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
      601 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
      661 cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg
      721 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
      781 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg
      841 ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg
      901 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc
      961 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc
     1021 gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
     1081 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc
     1141 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
     1201 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
     1261 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg
     1321 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
     1381 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
     1441 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
     1501 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
     1561 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
     1621 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
     1681 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc
     1741 ctcgaggaat tcactcctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     1801 aatgccctgg ctcacaaata ccactgagat ctgcatgagc tagcgccacc atggagggat
     1861 tcgacggttc agattttagt cctccagctg atttagttgg cgttgacggt gcagtcatgc
     1921 ggaacgtcgt tgacgtcacg ataaatggtg acgtcactgc tccgccgaaa gctgcgccaa
     1981 gaaaaagtga atcggtaaag aaagttcatt ggaatgatgt agaccaagga ccgagtgaaa
     2041 aaccagagac aagacaggag gaacgaatag atatacccga gatttcaggt ctatggtggg
     2101 gcgagaatga acatggagtg gacgatggga gaatggagat acctactact ggtgtaggtc
     2161 gcgtccagtt tcgtgtccga gcagtgattg atcatctagg gatgcgagtc tttggagtct
     2221 tcctaatttt cttggacatc atcctcatga tcattgatct cagtcttcca ggaaaaagtg
     2281 aatcttcaca atccttttat gacgggatgg ctttggctct ttcttgttat ttcatgctgg
     2341 atttaggatt aaggatattt gcctacgggc ccaagaattt cttcaccaac ccctgggagg
     2401 ttgctgatgg tttgattatc gttgtcacat tcgtcgtcac gatattttac actgtgttag
     2461 atgaatacgt gcaagaaaca ggagccgatg gtttggggca gttggttgtg ttggcccgtt
     2521 tgctgcgtgt ggttcgatta gcaagaatat tttattccca ccaacaaatg aaggcttcaa
     2581 gcagaagaac aatatcacag aacaagcgtc gaatggtgag caagggcgag gagctgttca
     2641 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     2701 tccgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca
     2761 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcgtgg
     2821 cctgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2881 ccgaaggcta cgtccaggag cgcaccatct ctttcaagga cgacggtacc tacaagaccc
     2941 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     3001 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca
     3061 acgtctatat cacggccgac aagcagaaga acggcatcaa ggctaacttc aagatccgcc
     3121 acaacgttga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     3181 gcgacggccc cgtgctgctg cccgacaacc actacctgag ccatcagtcc gccctgagca
     3241 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtaacc gccgccctcg
     3301 agtccggagg cgagatggtg tctaagggcg aagagctgat caaggaaaat atgcgtatga
     3361 aggtggtcat ggaaggttcg gtcaacggcc accaattcaa atgcacaggt gaaggagaag
     3421 gcaatccgta catgggaact caaaccatga ggatcaaagt catcgaggga ggacccctgc
     3481 catttgcctt tgacattctt gccacgtcgt tcatgtatgg cagccgtact tttatcaagt
     3541 acccgaaagg cattcctgat ttctttaaac agtcctttcc tgagggtttt acttgggaaa
     3601 gagttacgag atacgaagat ggtggagtcg tcaccgtcat gcaggacacc agccttgagg
     3661 atggctgtct cgtttaccac gtccaagtca gaggggtaaa ctttccctcc aatggtcccg
     3721 tgatgcagaa gaagaccaag ggttgggagc ctaatacaga gatgatgtat ccagcagatg
     3781 gtggtctgag gggatacact catatggcac tgaaagttga tggtggtggc catctgtctt
     3841 gctctttcgt aacaacttac aggtcaaaaa agaccgtcgg gaacatcaag atgcccggta
     3901 tccatgccgt tgatcaccgc ctggaaaggt tagaggaaag tgacaatgaa atgttcgtag
     3961 tacaacgcga acacgcagtt gccaagttcg ccgggcttgg tggtgggatg gacgagctgt
     4021 acaagtaata aaagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg
     4081 cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc
     4141 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     4201 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     4261 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     4321 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     4381 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     4441 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4501 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4561 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4621 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4681 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4741 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4801 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     4861 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4921 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4981 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     5041 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     5101 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     5161 ggcttttttg gaggcctagg cttttgcaaa aagctcccat gaccgagtac aagcccacgg
     5221 tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg
     5281 ccgactaccc cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg
     5341 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     5401 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     5461 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     5521 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     5581 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     5641 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     5701 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     5761 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgatt cgaaatgacc gaccaagcga
     5821 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5881 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5941 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     6001 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     6061 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     6121 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     6181 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     6241 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6301 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6361 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6421 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6481 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6541 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6601 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6661 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6721 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6781 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6841 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6901 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6961 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     7021 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca
     7081 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     7141 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     7201 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     7261 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     7321 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     7381 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     7441 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     7501 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     7561 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     7621 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7681 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7741 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7801 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7861 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7921 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7981 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     8041 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     8101 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     8161 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     8221 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     8281 tc
//
