LOCUS       Exported                7607 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  NanoLuc Gateway shuttle vector for N-terminal fusions.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1 myc-NL-GW
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7607)
  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   Molecular Systems Biology (2018) 14, e8071
REFERENCE   2  (bases 1 to 7607)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7607
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        103..482
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        483..686
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     637..657
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     731..750
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        731..749
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             775..804
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             805..1314
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     protein_bind    1321..1445
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1470..1500
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1554..2234
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(1620..1639)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2554..2859
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2738..2757
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2900..3024)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3066..3083)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3072..3296
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3342..3770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3429..3448)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3639..3660
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3779..3799)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3784..4113
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3964..4099
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4026..4045
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4180..4974
                     /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(4234..4253)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4844..4863
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5148..5269
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5185..5204)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5239..5258
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5318..5334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5318..5334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5331..5353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5342..5358
                     /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(5366..5396)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5411..5432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5549..5566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5720..6308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5800..5819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6479..7339)
                     /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     7102..7121
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7340..7444)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7519..7538)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
       61 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
      121 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      181 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      241 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      301 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      361 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      421 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      481 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      541 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      601 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      661 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
      721 ttatcgaaat taatacgact cactataggg agacccaagc tggctagcac catggaacag
      781 aaactgatct ctgaagaaga cctggtcttc acactcgaag atttcgttgg ggactggcga
      841 cagacagccg gctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgttt
      901 cagaatctcg gggtgtccgt aactccgatc caaaggattg tcctgagcgg tgaaaatggg
      961 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcggcga ccaaatgggc
     1021 cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc
     1081 ctgcactatg gcacactggt aatcgacggg gttacgccga acatgatcga ctatttcgga
     1141 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg
     1201 tggaacggca acaaaattat cgacgagcgc ctgatcaacc ccgacggctc cctgctgttc
     1261 cgagtaacca tcaacggagt gaccggctgg cggctgtgcg aacgcattct ggcgaagctt
     1321 acaagtttgt acaaaaaagc tgaacgagaa acgtaaaatg atataaatat caatatatta
     1381 aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca
     1441 ctatggcggc cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg
     1501 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
     1561 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     1621 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     1681 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     1741 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
     1801 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     1861 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     1921 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     1981 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     2041 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     2101 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     2161 gaatgcttaa tgaattacaa cagtactgcg atgagtggca ggcggggcgt aatctagagg
     2221 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     2281 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     2341 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     2401 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     2461 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     2521 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     2581 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     2641 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt cccccgtgaa
     2701 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     2761 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     2821 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     2881 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt
     2941 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     3001 ttcagctttc ttgtacaaag tggtgtaact cgagtctaga gggcccgttt aaacccgctg
     3061 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     3121 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     3181 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     3241 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     3301 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     3361 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3421 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3481 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3541 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3601 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3661 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3721 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3781 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3841 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3901 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3961 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4021 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     4081 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     4141 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     4201 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4261 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4321 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     4381 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4441 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4501 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4561 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4621 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4681 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     4741 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4801 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4861 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4921 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4981 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     5041 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     5101 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     5161 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5221 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     5281 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5341 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5401 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5461 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5521 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5581 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5641 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5701 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5761 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5821 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5881 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5941 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6001 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6061 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6121 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6181 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6241 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6301 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6361 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6421 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6481 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6541 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6601 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6661 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6721 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6781 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6841 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6901 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6961 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7021 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7081 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7141 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7201 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7261 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7321 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7381 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7441 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     7501 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     7561 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaattt
//
