LOCUS       sequence-214025-        6997 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 6997)
  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 6997)
  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..6997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             909..1082
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             1083..1256
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNGAQAPK"
     CDS             1598..1609
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    1980..2004
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2007..2016
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2013..2525
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     protein_bind    complement(2527..2551)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2593..2610)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2599..2823
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2869..3297
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2956..2975)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3166..3187
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3306..3326)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3311..3640
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3491..3626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3553..3572
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3707..4501
                     /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(3761..3780)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4371..4390
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4675..4796
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4712..4731)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4766..4785
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4845..4861)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4845..4861)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4858..4880)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4869..4885
                     /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(4893..4899,4900..4917,4918..4923))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4938..4959
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5076..5093)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5247..5832)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5327..5346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6003..6794,6795..6863))
                     /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     6626..6645
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6864..6968)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
      901 gcaccatggt ggacaacaaa ttcaacaaag aacaacaaaa cgcgttctat gagatcttac
      961 atttacctaa cttaaacgaa gaacaacgaa acgccttcat ccaaagttta aaagatgacc
     1021 caagccaaag cgctaacctt ttagcagaag ctaaaaagct aaatgatgct caggcgccga
     1081 aagtagacaa caaattcaac aaagaacaac aaaacgcgtt ctatgagatc ttacatttac
     1141 ctaacttaaa cgaagaacaa cgaaacgcct tcatccaaag tttaaaagat gacccaagcc
     1201 aaagcgctaa ccttttagca gaagctaaaa agctaaatgg tgctcaggcg ccgaaagtag
     1261 acgcgaactc ggaattcggc tcgggctcgg tatcgaaagg tgaagagctg tttactggcg
     1321 tcgtgcccat tttggtggaa cttgacggtg acgtgaacgg tcacaaattc tcagtctcgg
     1381 gagaaggcga gggggacgcg acatatggga aactcacact gaagttcatc tgcacgactg
     1441 gaaagctgcc tgtcccttgg cctacactcg tcacgacgtt cggttatggt ttgatgtgtt
     1501 tcgcaagata tcctgaccac atgaaacagc atgatttctt taagagcgcc atgcccgaag
     1561 gttatgtcca ggagaggact atcttcttta aggacgatgg gaactacaag actcgggcag
     1621 aggtgaaatt tgagggggac acgcttgtga atagaatcga actcaaaggc atcgatttca
     1681 aggaggatgg aaacattctc ggacacaaac ttgagtacaa ttacaacagc cacaacgtgt
     1741 atatcatggc ggacaaacag aagaatggaa tcaaagtgaa cttcaagatc agacacaata
     1801 tcgaagatgg gtcagtgcag ctggctgatc attaccagca gaatacgccc atcggggacg
     1861 gtccagtctt gttgccggat aaccactacc tttcatacca gtcgaagttg tccaaagacc
     1921 ccaacgagaa gagggatcac atggtgcttc ttgaatttgt cacagccgct ggtaagctta
     1981 caagtttgta caaaaaagca ggcttcgcca ccatggtctt cacactcgaa gatttcgttg
     2041 gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt
     2101 ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt gtcctgagcg
     2161 gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt ctgagcggcg
     2221 accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat gatcatcact
     2281 ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg aacatgatcg
     2341 actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa
     2401 cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac cccgacggct
     2461 ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc gaacgcattc
     2521 tggcggaccc agctttcttg tacaaagtgg tgtaactcga gtctagaggg cccgtttaaa
     2581 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2641 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2701 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2761 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2821 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2881 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2941 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3001 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3061 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3121 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3181 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3241 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3301 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3361 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3421 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3481 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3541 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3601 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3661 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3721 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3781 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3841 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3901 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3961 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4021 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4081 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4141 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4201 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     4261 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4321 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4381 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4441 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4501 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4561 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4621 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4681 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4741 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4801 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4861 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4921 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4981 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5041 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5101 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5161 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5221 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5281 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5341 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5401 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5461 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5521 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5581 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5641 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5701 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5761 aaacaaacca ccgctggtag cggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5821 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5881 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5941 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6001 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6061 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6121 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6181 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6241 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6301 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6361 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6421 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6481 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6541 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6601 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6661 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6721 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6781 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6841 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6901 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6961 gttccgcgca catttccccg aaaagtgcca cctgacg
//
