LOCUS       Exported                7720 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  N-terminal EGFP tag. Gateway destination vector for mammalian 
            expression..
ACCESSION   .
VERSION     .
KEYWORDS    pDEST-CMV-N-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7720)
  AUTHORS   Agrotis A, Pengo N, Burden JJ, Ketteler R
  TITLE     Redundancy of human ATG4 protease isoforms in autophagy and 
            LC3/GABARAP processing revealed in cells.
  JOURNAL   Autophagy. 2019 Jan 21. doi: 10.1080/15548627.2019.1569925.
  PUBMED    30661429
REFERENCE   2  (bases 1 to 7720)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7720
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     regulatory      852..861
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             858..1574
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(903..924)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1164..1183)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1511..1532
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1596..1720
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1745..1775
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1829..2488
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1895..1914)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2809..3114
                     /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
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2993..3012
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3155..3279)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3311..3328)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3317..3541
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3587..4015
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3674..3693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3884..3905
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4024..4044)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4029..4358
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4209..4344
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4271..4290
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4425..5219
                     /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(4479..4498)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5089..5108
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5393..5514
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5430..5449)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5484..5503
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5563..5579)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5563..5579)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5576..5598)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5587..5603
                     /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(5611..5641)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5656..5677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5794..5811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5965..6553)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6045..6064)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6724..7584)
                     /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     7347..7366
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7585..7689)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 gcagagctcg tttagtgaac cgtcagaatt
      841 gatctggtac cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      901 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      961 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1021 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1081 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1141 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1201 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1261 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1321 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1381 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1441 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1501 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact cacggcatgg
     1561 acgagctcta caaaagcggt tccggaccgg atcaaacaag tttgtacaaa aaagctgaac
     1621 gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga
     1681 ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat taggcacccc
     1741 aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccgtc
     1801 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt
     1861 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg
     1921 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa
     1981 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc
     2041 ggaattccgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg
     2101 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga
     2161 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct
     2221 ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt
     2281 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt
     2341 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
     2401 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta
     2461 ctgcgatgag tggcagggcg gggcgtaaac gcgtggatcc ggcttactaa aagccagata
     2521 acagtatgcg tatttgcgcg ctcgcgaacc ggtgtatacc cgaagtatgt caaaaagagg
     2581 tatgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag
     2641 gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc
     2701 gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt
     2761 ttattgaaat gaacggctct tttgctgacg agaacagggg ctggtgaaat gcagtttaag
     2821 gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt
     2881 attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat
     2941 aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg
     3001 accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc
     3061 caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca
     3121 ggctccctta tacacagcca gtctgcaggt cgaccatagt gactggatat gttgtgtttt
     3181 acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc
     3241 attttacgtt tctcgttcag ctttcttgta caaagtggtg ataattaatt aagataaacc
     3301 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3361 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3421 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3481 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3541 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3601 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3661 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3721 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3781 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3841 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3901 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3961 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     4021 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     4081 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     4141 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     4201 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4261 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     4321 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     4381 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4441 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4501 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4561 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4621 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4681 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4741 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4801 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4861 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4921 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4981 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     5041 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     5101 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     5161 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     5221 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     5281 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5341 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     5401 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5461 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5521 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5581 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5641 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5701 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5761 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5821 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5881 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5941 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6001 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6061 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6121 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6181 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6241 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6301 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6361 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6421 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6481 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6541 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6601 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6661 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6721 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6781 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6841 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6901 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6961 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7021 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7081 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7141 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7201 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7261 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7321 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7381 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7441 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7501 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7561 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7621 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7681 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
