LOCUS       sequence-8526-ex        6932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pFlag-CMV-2 converted Gateway Destination vector for expressing Flag
            tagged fusion proteins in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6932)
  AUTHORS   Guo F, Chiang MY, Wang Y, Zhang YZ
  TITLE     An in vitro recombination method to convert restriction- and 
            ligation-independent expression vectors.
  JOURNAL   Biotechnol J. 2008 Mar . 3(3):370-7.
  PUBMED    18064608
REFERENCE   2  (bases 1 to 6932)
  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: "Biotechnol 
            J. 2008 Mar . 3(3):370-7."
COMMENT     Sequence Label: pEZYflag
FEATURES             Location/Qualifiers
     source          1..6932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             935..958
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    986..1110
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1539..1844)
                     /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     complement(1641..1660)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2189..2848)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2763..2782
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2849..2951)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(3129..3253)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3273..3895
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3472..3491)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3919..3939)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3924..4253
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4104..4239
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4291..4310)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4292..4310)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4324..4340)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4324..4340)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4337..4359)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4348..4364
                     /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(4372..4378,4379..4396,4397..4402))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4417..4438
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4555..4572)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4726..5314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4806..4825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5485..6276,6277..6345))
                     /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     6108..6127
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6346..6450)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6477..6905
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6564..6583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6774..6795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attgatctac catggactac aaagacgatg acgacaagct
      961 tgcggccgcc atgcaattga ttaatacaag tttgtacaaa aaagctgaac gagaaacgta
     1021 aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata
     1081 ctgtaaaaca caacatatcc agtcactatg aatcaactac ttagatggta ttagtgacct
     1141 gtagtcgacc gacagccttc caaatgttct tcgggtgatg ctgccaactt agtcgaccga
     1201 cagccttcca aatgttcttc tcaaacggaa tcgtcgtatc cagcctactc gctattgtcc
     1261 tcaatgccgt attaaatcat aaaaagaaat aagaaaaaga ggtgcgagcc tcttttttgt
     1321 gtgacaaaat aaaaacatct acctattcat atacgctagt gtcatagtcc tgaaaatcat
     1381 ctgcatcaag aacaatttca caactcttat acttttctct tacaagtcgt tcggcttcat
     1441 ctggattttc agcctctata cttactaaac gtgataaagt ttctgtaatt tctactgtat
     1501 cgacctgcag actggctgtg tataagggag cctgacattt atattcccca gaacatcagg
     1561 ttaatggcgt ttttgatgtc attttcgcgg tggctgagat cagccacttc ttccccgata
     1621 acggagaccg gcacactggc catatcggtg gtcatcatgc gccagctttc atccccgata
     1681 tgcaccaccg ggtaaagttc acgggagact ttatctgaca gcagacgtgc actggccagg
     1741 gggatcacca tccgtcgccc gggcgtgtca ataatatcac tctgtacatc cacaaacaga
     1801 cgataacggc tctctctttt ataggtgtaa accttaaact gcatttcacc agcccctgtt
     1861 ctcgtcagca aaagagccgt tcatttcaat aaaccgggcg acctcagcca tcccttcctg
     1921 attttccgct ttccagcgtt cggcacgcag acgacgggct tcattctgca tggttgtgct
     1981 taccagaccg gagatattga catcatatat gccttgagca actgatagct gtcgctgtca
     2041 actgtcactg taatacgctg cttcatagca tacctctttt tgacatactt cgggtataca
     2101 tatcagtata tattcttata ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg
     2161 cttttagtaa gccggatcca cgcggcgttt acgccccgcc ctgccactca tcgcagtact
     2221 gttgtaattc attaagcatt ctgccgacat ggaagccatc acagacggca tgatgaacct
     2281 gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa
     2341 cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc
     2401 agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt
     2461 tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt
     2521 ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag
     2581 ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat
     2641 gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt
     2701 tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt
     2761 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg
     2821 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata
     2881 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     2941 cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag
     3001 ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc
     3061 aaagtgcgtc gggtgatgct gccaacttag tcgactacag gtcactaata ccatctaagt
     3121 agttgattca tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat
     3181 gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcagctttct
     3241 tgtacaaagt ggtcctcgag gtaccggatc ccgggtggca tccctgtgac ccctccccag
     3301 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat
     3361 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg
     3421 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg
     3481 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc
     3541 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc
     3601 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct
     3661 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg
     3721 cgtgaaccac tgctcccttc cctgtccttc tgattttaaa ataactatac cagcaggagg
     3781 acgtccagac acagcatagg ctacctggcc atgcccaacc ggtgggacat ttgagttgct
     3841 tgcttggcac tgtcctctca tgcgttgggt ccactcagta gatgcctgtt gaattgggta
     3901 cgcggccagc ttggctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3961 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     4021 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4081 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4141 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     4201 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     4261 tcgaggaact gaaaaaccag aaagttaatt ccctatagtg agtcgtatta aattcgtaat
     4321 catgtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4381 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4441 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4501 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4561 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4621 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4681 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4741 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4801 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4861 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4921 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4981 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5041 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5101 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5161 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5221 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5281 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5341 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5401 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5461 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5521 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5581 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5641 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5701 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5761 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5821 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5881 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5941 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6001 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6061 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6121 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6181 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6241 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6301 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6361 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6421 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc
     6481 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     6541 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     6601 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     6661 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     6721 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     6781 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     6841 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     6901 gaattttaac aaaatattaa cgcttacaat tt
//
