LOCUS       sequence-99628-e        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains FLAG epitope for immunofluorescence detection of resorufin 
            ligase for mammalian cell expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6558)
  AUTHORS   Liu DS, Nivon LG, Richter F, Goldman PJ, Deerinck TJ, Yao JZ, 
            Richardson D, Phipps WS, Ye AZ, Ellisman MH, Drennan CL, Baker D, 
            Ting AY
  TITLE     Computational design of a red fluorophore ligase for site-specific 
            protein labeling in living cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Oct 28;111(43):E4551-9. doi: 
            10.1073/pnas.1404736111. Epub 2014 Oct 13.
  PUBMED    25313043
REFERENCE   2  (bases 1 to 6558)
  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"; doi: 
            "10.1073/pnas.1404736111"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2014-10-28- 28"; volume: "111"; issue: "43"; pages: "E4551-9"
COMMENT     Sequence Label: pcDNA3-FLAG-LplA(AAG)
FEATURES             Location/Qualifiers
     source          1..6558
                     /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"
     CDS             1009..1032
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2110..2128)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2111..2128)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2148..2165)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2154..2378
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2424..2852
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2511..2530)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2721..2742
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2861..2881)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2866..3196
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3047..3182
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3109..3128
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3263..4057
                     /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(3317..3336)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3927..3946
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4231..4352
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4268..4287)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4322..4341
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4401..4417)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4401..4417)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4414..4436)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4425..4441
                     /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(4449..4455,4456..4473,4474..4479))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4494..4515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4632..4649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4803..5391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4883..4902)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5562..6353,6354..6422))
                     /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     6185..6204
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6423..6527)
                     /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 aggatccaga ctacaaggat
     1021 gacgacgata agatgagcac cctgagactg ctgatcagcg acagctacga cccctggttc
     1081 aacctggccg tggccgagtg catcttcaga cagatgcccg ccacccagag agtgctgttc
     1141 ctgtggagaa acgccgacac cgtggtgatc ggcagagccc agaacccctg gaaggagtgc
     1201 aacaccagaa gaatggagga ggacaacgtg agactggcca gaagaagcag cggcggcggc
     1261 gccgtgttcc acgacctggg caacacctgc ttcaccttca tggccggcaa gcccgagtac
     1321 gacaagacca tcagcaccag catcgtgctg aacgccctga acgccctggg cgtgagcgcc
     1381 gaggccagcg gcagaaacga cctggtggtg aagaccgtgg agggcgacag aaaggtgagc
     1441 ggcagcgcct acagagagac caaggacaga ggcgcccacg gcggcaccct gctgctgaac
     1501 gccgacctga gcagactggc caactacctg aaccccgaca agaagaagct ggccgccaag
     1561 ggcatcacca gcgtgagaag cagagtgacc aacctgaccg agctgctgcc cggcatcacc
     1621 cacgagcagg tgtgcgaggc catcaccgag gccttcttcg cccactacgg cgagagagtg
     1681 gaggccgaga tcatcagccc caacaagacc cccgacctgc ccaacttcgc cgagaccttc
     1741 gccagacaga gcagctggga gtggaacttc ggccaggccc ccgccttcag ccacctgctg
     1801 gacgagagat tcacctgggg cggcgtggag ctgcacttcg acgtggagaa gggccacatc
     1861 accagagccc aggtgttcac cgacagcctg aaccccgccc ccctggaggc cctggccggc
     1921 agactgcagg gctgcctgta cagagccgac atgctgcagc aggagtgcga ggccctgctg
     1981 gtggacttcc ccgagcagga gaaggagctg agagagctga gcgcctggat ggccggcgcc
     2041 gtgagataag aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga
     2101 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     2161 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2221 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2281 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2341 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2401 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2461 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2521 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2581 catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2641 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2701 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2761 ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa
     2821 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2881 tgtggaaagt ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta
     2941 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3001 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3061 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3121 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3181 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     3241 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3301 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3361 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3421 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     3481 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3541 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3601 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3661 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3721 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3781 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3841 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3901 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3961 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4021 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4081 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4141 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4201 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4261 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4321 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4381 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4441 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4501 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4561 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4621 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4681 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4741 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4801 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4861 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4921 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4981 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5041 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5101 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5161 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5221 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     5281 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5341 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5401 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5461 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5521 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5581 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5641 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5701 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5761 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5821 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5881 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5941 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6001 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6061 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6121 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6181 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6241 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6301 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6361 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6421 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6481 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6541 aaagtgccac ctgacgtc
//
