LOCUS       sequence-218202-        6990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ProteinA-mCitrine-NanoLuc hybrid-fusion control expression vector 
            (positive control).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6990)
  AUTHORS   Trepte P, Kruse S, Kostova S, Hoffmann S, Buntru A, Tempelmeier A, 
            Secker C, Diez L, Schulz A, Klockmeier K, Zenkner M, Golusik S, Rau 
            K, Schnoegl S, Garner CC, Wanker EE
  TITLE     LuTHy: a double-readout bioluminescence-based two-hybrid technology 
            for quantitative mapping of protein-protein interactions in 
            mammalian cells.
  JOURNAL   Mol Syst Biol. 2018 Jul 11;14(7):e8071. doi: 10.15252/msb.20178071.
  PUBMED    29997244
REFERENCE   2  (bases 1 to 6990)
  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: "Mol Syst 
            Biol."; date: "2018-07-11"; volume: "14(7):e8071. doi"; pages: " 
            10.15252/msb.20178071"
COMMENT     Sequence Label: pcDNA3.1 PA-mCit-NL
FEATURES             Location/Qualifiers
     source          1..6990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        122..501
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        502..705
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     656..676
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     750..769
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        750..768
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             794..967
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             968..1141
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNGAQAPK"
     CDS             1483..1494
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    1865..1889
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1892..1901
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1898..2410
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     protein_bind    complement(2412..2436)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2478..2495)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2484..2595
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2744..3172
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2831..2850)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3041..3062
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3181..3201)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3186..3515
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3366..3501
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3428..3447
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3582..4376
                     /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(3636..3655)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4246..4265
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4550..4671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4587..4606)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4641..4660
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4720..4736)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4720..4736)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4733..4755)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4744..4760
                     /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(4768..4774,4775..4792,4793..4798))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4813..4834
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4951..4968)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5122..5710)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5202..5221)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5881..6672,6673..6741))
                     /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     6504..6523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6742..6846)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6921..6940)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
       61 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
      121 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      181 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      241 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      301 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      361 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      421 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      481 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      541 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      601 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      661 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
      721 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
      781 ggctagcacc atggtggaca acaaattcaa caaagaacaa caaaacgcgt tctatgagat
      841 cttacattta cctaacttaa acgaagaaca acgaaacgcc ttcatccaaa gtttaaaaga
      901 tgacccaagc caaagcgcta accttttagc agaagctaaa aagctaaatg atgctcaggc
      961 gccgaaagta gacaacaaat tcaacaaaga acaacaaaac gcgttctatg agatcttaca
     1021 tttacctaac ttaaacgaag aacaacgaaa cgccttcatc caaagtttaa aagatgaccc
     1081 aagccaaagc gctaaccttt tagcagaagc taaaaagcta aatggtgctc aggcgccgaa
     1141 agtagacgcg aactcggaat tcggctcggg ctcggtatcg aaaggtgaag agctgtttac
     1201 tggcgtcgtg cccattttgg tggaacttga cggtgacgtg aacggtcaca aattctcagt
     1261 ctcgggagaa ggcgaggggg acgcgacata tgggaaactc acactgaagt tcatctgcac
     1321 gactggaaag ctgcctgtcc cttggcctac actcgtcacg acgttcggtt atggtttgat
     1381 gtgtttcgca agatatcctg accacatgaa acagcatgat ttctttaaga gcgccatgcc
     1441 cgaaggttat gtccaggaga ggactatctt ctttaaggac gatgggaact acaagactcg
     1501 ggcagaggtg aaatttgagg gggacacgct tgtgaataga atcgaactca aaggcatcga
     1561 tttcaaggag gatggaaaca ttctcggaca caaacttgag tacaattaca acagccacaa
     1621 cgtgtatatc atggcggaca aacagaagaa tggaatcaaa gtgaacttca agatcagaca
     1681 caatatcgaa gatgggtcag tgcagctggc tgatcattac cagcagaata cgcccatcgg
     1741 ggacggtcca gtcttgttgc cggataacca ctacctttca taccagtcga agttgtccaa
     1801 agaccccaac gagaagaggg atcacatggt gcttcttgaa tttgtcacag ccgctggtaa
     1861 gcttacaagt ttgtacaaaa aagcaggctt cgccaccatg gtcttcacac tcgaagattt
     1921 cgttggggac tggcgacaga cagccggcta caacctggac caagtccttg aacagggagg
     1981 tgtgtccagt ttgtttcaga atctcggggt gtccgtaact ccgatccaaa ggattgtcct
     2041 gagcggtgaa aatgggctga agatcgacat ccatgtcatc atcccgtatg aaggtctgag
     2101 cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca
     2161 tcactttaag gtgatcctgc actatggcac actggtaatc gacggggtta cgccgaacat
     2221 gatcgactat ttcggacggc cgtatgaagg catcgccgtg ttcgacggca aaaagatcac
     2281 tgtaacaggg accctgtgga acggcaacaa aattatcgac gagcgcctga tcaaccccga
     2341 cggctccctg ctgttccgag taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg
     2401 cattctggcg gacccagctt tcttgtacaa agtggtgtaa ctcgagtcta gagggcccgt
     2461 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2521 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2581 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2641 gcaggacagc aagggggagg attgggcagg catgctgggg atgcggtggg ctctatggct
     2701 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2761 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2821 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2881 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2941 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3001 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3061 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3121 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3181 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3241 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3301 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3361 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3421 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3481 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3541 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3601 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3661 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3721 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3781 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3841 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3901 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3961 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4021 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4081 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4141 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4201 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4261 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4321 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4381 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4441 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4501 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4561 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4621 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4681 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4741 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4801 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4861 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4921 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4981 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5041 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5101 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5161 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5221 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5281 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5341 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5401 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5461 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5521 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5581 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5641 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5701 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5761 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5821 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5881 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5941 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6001 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6061 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6121 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6181 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6241 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6301 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6361 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6421 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6481 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6541 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6601 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6661 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6721 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6781 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6841 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     6901 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     6961 ctccctgctt gtgtgttgga ggtcgctgag
//
