LOCUS       sequence-51813-e        7398 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 7398)
  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 7398)
  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-AKAR2-CR
FEATURES             Location/Qualifiers
     source          1..7398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1054..1075)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1662..1683
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2215..2925
                     /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"
     promoter        complement(2950..2968)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2951..2968)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2988..3005)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2994..3218
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3264..3692
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3351..3370)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3561..3582
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3701..3721)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3706..4036
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3887..4022
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3949..3968
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4103..4897
                     /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(4157..4176)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4767..4786
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5071..5192
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5108..5127)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5162..5181
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5241..5257)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5241..5257)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5254..5276)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5265..5281
                     /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(5289..5295,5296..5313,5314..5319))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5334..5355
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5472..5489)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5643..6231)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5723..5742)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6402..7193,7194..7262))
                     /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     7025..7044
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7263..7367)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatccgat ggtgagcaag
     1021 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1081 ggccacaagt tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg caagctgacc
     1141 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1201 ttcggctacg gcgtggcctg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1261 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcttt caaggacgac
     1321 ggtacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1381 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1441 aacttcaaca gccacaacgt ctatatcacg gccgacaagc agaagaacgg catcaaggct
     1501 aacttcaaga tccgccacaa cgttgaggac ggcagcgtgc agctcgccga ccactaccag
     1561 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagccat
     1621 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1681 gtgaccgccg cccgcatgca taagttttct caagaacaga tcggcgaaaa cattgtgtgc
     1741 agggtcattt gtaccacggg tcaaattccc atccgagatt tgtcagctga tatttcacaa
     1801 gtgcttaagg aaaaacgatc cataaagaaa gtttggacat ttggtagaaa cccagcctgt
     1861 gactatcatt taggaaacat ttcaagactg tcaaataagc atttccaaat actactagga
     1921 gaagacggta accttttatt gaatgacatt tccactaatg ggacctggtt aaatgggcaa
     1981 aaagtcgaga agaacagcaa tcagttactg tctcaaggtg atgaaataac cgttggtgta
     2041 ggcgtggaat cagatatttt atctctggtc attttcataa acgacaaatt taagcagtgc
     2101 ctcgagcaga acaaagttga tcgctctgca ggtaagccag gcagcggcga gggcagcacc
     2161 aagggcctgc gtcgcgccac cctggtggac ggcggcaccg gcggcagcga gctcatggtg
     2221 tctaagggcg aagagctgat caaggaaaat atgcgtatga aggtggtcat ggaaggttcg
     2281 gtcaacggcc accaattcaa atgcacaggt gaaggagaag gcaatccgta catgggaact
     2341 caaaccatga ggatcaaagt catcgaggga ggacccctgc catttgcctt tgacattctt
     2401 gccacgtcgt tcatgtatgg cagccgtact tttatcaagt acccgaaagg cattcctgat
     2461 ttctttaaac agtcctttcc tgagggtttt acttgggaaa gagttacgag atacgaagat
     2521 ggtggagtcg tcaccgtcat gcaggacacc agccttgagg atggctgtct cgtttaccac
     2581 gtccaagtca gaggggtaaa ctttccctcc aatggtcccg tgatgcagaa gaagaccaag
     2641 ggttgggagc ctaatacaga gatgatgtat ccagcagatg gtggtctgag gggatacact
     2701 catatggcac tgaaagttga tggtggtggc catctgtctt gctctttcgt aacaacttac
     2761 aggtcaaaaa agaccgtcgg gaacatcaag atgcccggta tccatgccgt tgatcaccgc
     2821 ctggaaaggt tagaggaaag tgacaatgaa atgttcgtag tacaacgcga acacgcagtt
     2881 gccaagttcg ccgggcttgg tggtgggatg gacgagctgt acaagtaaga attctctaga
     2941 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     3001 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3061 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3121 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3181 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3241 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3301 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3361 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3421 catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3481 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3541 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3601 ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa
     3661 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3721 tgtggaaagt ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta
     3781 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3841 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3901 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3961 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4021 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     4081 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4141 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4201 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4261 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4321 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4381 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4441 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4501 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4561 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4621 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4681 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4741 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4801 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4861 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4921 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4981 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5041 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     5101 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5161 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5221 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5281 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5341 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5401 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5461 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5521 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5581 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5641 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5701 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5761 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5821 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5881 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5941 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6001 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6061 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     6121 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6181 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6241 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6301 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6361 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6421 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6481 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6541 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6601 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6661 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6721 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6781 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6841 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6901 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6961 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7021 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7081 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7141 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7201 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7261 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7321 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7381 aaagtgccac ctgacgtc
//
