LOCUS       sequence-361454-        6035 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Gateway converted, destination vector with N-terminal Flag tag. For 
            mammalian expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6035)
  AUTHORS   Panza E, Marini M, Pecci A, Giacopelli F, Bozzi V, Seri M, Balduini 
            C, Ravazzolo R
  TITLE     Transfection of the mutant MYH9 cDNA reproduces the most typical 
            cellular phenotype of MYH9-related disease in different cell lines.
  JOURNAL   Pathogenetics. 2008 Dec 1;1(1):5. doi: 10.1186/1755-8417-1-5.
  PUBMED    19046415
REFERENCE   2  (bases 1 to 6035)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Pathogenetics."; date: "2008-12-1"; volume: "1(1):5. doi"; pages: "
            10.1186/1755-8417-1-5"
COMMENT     Sequence Label: pDEST-FLAG
FEATURES             Location/Qualifiers
     source          1..6035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        67..370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        371..574
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     525..545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        620..638
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      673..682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             682..705
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    745..869
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(894..899,900..917,918..924)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             978..1637
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1044..1063)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1979..2284
                     /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     2163..2182
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2325..2449)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2464..2480)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2465..2481)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2536..2555)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2537..2555)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2829..2950
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2866..2885)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2920..2939
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2957..3412)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3094..3115)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3306..3325
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3439..3543
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3545..3902
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3545..3736
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3568..3588)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3753..3888
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3937..4731
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3937..4731
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3991..4010)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4601..4620
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4919..4938)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4963..5010
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5339..5927
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5828..5847
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
       61 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      121 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      181 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      241 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      301 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      361 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      421 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      481 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      541 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      601 gcgattacgc caagctcgaa attaaccctc actaaaggga acaaaagctg gagctccacc
      661 gcggtggcgg ccgccaccat ggattacaag gatgacgacg ataagagccc gggcggatcc
      721 cccgggctgc aggaattcga tatcacaagt ttgtacaaaa aagctgaacg agaaacgtaa
      781 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
      841 tgtaaaacac aacatatcca gtcactatgg cggccgcatt aggcacccca ggctttacac
      901 tttatgcttc cggctcgtat aatgtgtgga ttttgagtta ggatccgtcg agattttcag
      961 gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc
     1021 aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc
     1081 agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt
     1141 tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta
     1201 tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt
     1261 tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc
     1321 agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc
     1381 ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca
     1441 gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca
     1501 aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg
     1561 tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt
     1621 ggcagggcgg ggcgtaaacg cgtggatccg gcttactaaa agccagataa cagtatgcgt
     1681 atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt
     1741 caaaaagagg tatgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca
     1801 gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat
     1861 gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag
     1921 gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacagggg ctggtgaaat
     1981 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     2041 gagtgatatt attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct
     2101 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     2161 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     2221 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     2281 ataaatgtca ggctccctta tacacagcca gtctgcaggt cgaccatagt gactggatat
     2341 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     2401 tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggtg atatcaagct
     2461 tatcgatacc gtcgacctcg agggggggcc cggtacctta attaattaag gtaccaggta
     2521 agtgtaccca attcgcccta tagtgagtcg tattacaatt cactcgatcg cccttcccaa
     2581 cagttgcgca gcctgaatgg cgaatggaga tccaattttt aagtgtataa tgtgttaaac
     2641 tactgattct aattgtttgt gtattttaga ttcacagtcc caaggctcat ttcaggcccc
     2701 tcagtcctca cagtctgttc atgatcataa tcagccatac cacatttgta gaggttttac
     2761 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     2821 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     2881 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     2941 atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     3001 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     3061 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     3121 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     3181 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     3241 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     3301 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     3361 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     3421 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3481 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3541 aatcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3601 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3661 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3721 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3781 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3841 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3901 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3961 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4021 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4081 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     4141 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4201 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4261 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4321 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4381 gaagccggtc ttgtcgatca ggatgatctg gacgaagaac atcaggggct cgcgccagcc
     4441 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     4501 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4561 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4621 gctgaagaac ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4681 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4741 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4801 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4861 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     4921 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     4981 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     5041 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     5101 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     5161 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     5221 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5281 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5341 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5401 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5461 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5521 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5581 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5641 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5701 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5761 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5821 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5881 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5941 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6001 tatcccctga ttctgtggat aaccgtatta ccgcc
//
