LOCUS       sequence-229536-        6931 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 6931)
  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 6931)
  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..6931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     56..78
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     70..87
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     71..87
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        248..627
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        628..831
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     782..802
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             865..888
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    916..1040
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1469..1774)
                     /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(1571..1590)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2094..2777)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2692..2711
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2778..2880)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(3058..3182)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3202..3824
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3401..3420)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3848..3868)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3853..4182
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4033..4168
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4220..4239)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4221..4239)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4253..4269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4253..4269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4266..4288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4277..4293
                     /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(4301..4307,4308..4325,4326..4331))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4346..4367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4484..4501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4655..5243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4735..4754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5414..6205,6206..6274))
                     /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     6037..6056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6275..6379)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6406..6861
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6493..6512)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6703..6724
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
       61 tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa tcaatattgg
      121 caattagcca tattagtcat tggttatata gcataaatca atattggcta ttggccattg
      181 catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc aatatgaccg
      241 ccatgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
      301 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      361 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      421 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      481 gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg
      541 cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc
      601 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac caatgggcgt
      661 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      721 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg
      781 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg
      841 aaccgtcaga attgatctac catggactac aaagacgatg acgacaagct tgcggccgcc
      901 atgcaattga ttaatacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata
      961 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca
     1021 caacatatcc agtcactatg aatcaactac ttagatggta ttagtgacct gtagtcgacc
     1081 gacagccttc caaatgttct tcgggtgatg ctgccaactt agtcgaccga cagccttcca
     1141 aatgttcttc tcaaacggaa tcgtcgtatc cagcctactc gctattgtcc tcaatgccgt
     1201 attaaatcat aaaaagaaat aagaaaaaga ggtgcgagcc tcttttttgt gtgacaaaat
     1261 aaaaacatct acctattcat atacgctagt gtcatagtcc tgaaaatcat ctgcatcaag
     1321 aacaatttca caactcttat acttttctct tacaagtcgt tcggcttcat ctggattttc
     1381 agcctctata cttactaaac gtgataaagt ttctgtaatt tctactgtat cgacctgcag
     1441 actggctgtg tataagggag cctgacattt atattcccca gaacatcagg ttaatggcgt
     1501 ttttgatgtc attttcgcgg tggctgagat cagccacttc ttccccgata acggagaccg
     1561 gcacactggc catatcggtg gtcatcatgc gccagctttc atccccgata tgcaccaccg
     1621 ggtaaagttc acgggagact ttatctgaca gcagacgtgc actggccagg gggatcacca
     1681 tccgtcgccc gggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc
     1741 tctctctttt ataggtgtaa accttaaact gcatttcacc agcccctgtt ctcgtcagca
     1801 aaagagccgt tcatttcaat aaaccgggcg acctcagcca tcccttcctg attttccgct
     1861 ttccagcgtt cggcacgcag acgacgggct tcattctgca tggttgtgct taccagaccg
     1921 gagatattga catcatatat gccttgagca actgatagct gtcgctgtca actgtcactg
     1981 taatacgctg cttcatagca tacctctttt tgacatactt cgggtataca tatcagtata
     2041 tattcttata ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg cttttagtaa
     2101 gccggatcca cgcggcgttt acgccccccc tgccactcat cgcagtactg ttgtaattca
     2161 ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc
     2221 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     2281 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     2341 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     2401 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     2461 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     2521 tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc
     2581 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     2641 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     2701 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     2761 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat
     2821 acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca
     2881 acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg gacaccagga
     2941 tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca aagtgcgtcg
     3001 ggtgatgctg ccaacttagt cgactacagg tcactaatac catctaagta gttgattcat
     3061 agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa
     3121 tttaatatat tgatatttat atcattttac gtttctcgtt cagctttctt gtacaaagtg
     3181 gtcctcgagg taccggatcc cgggtggcat ccctgtgacc cctccccagt gcctctcctg
     3241 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc
     3301 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc
     3361 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg
     3421 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc
     3481 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt
     3541 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa
     3601 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact
     3661 gctcccttcc ctgtccttct gattttaaaa taactatacc agcaggagga cgtccagaca
     3721 cagcataggc tacctggcca tgcccaaccg gtgggacatt tgagttgctt gcttggcact
     3781 gtcctctcat gcgttgggtc cactcagtag atgcctgttg aattgggtac gcggccagct
     3841 tggctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3901 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3961 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4021 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4081 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4141 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctcct cgaggaactg
     4201 aaaaaccaga aagttaattc cctatagtga gtcgtattaa attcgtaatc atgtcatagc
     4261 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4321 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4381 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4441 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4501 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4561 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4621 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4681 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4741 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4801 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4861 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4921 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4981 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5041 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5101 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5161 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5221 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5281 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5341 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5401 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5461 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5521 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5581 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5641 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5701 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5761 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5821 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5881 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5941 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6001 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6061 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6121 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6181 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6241 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6301 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6361 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg
     6421 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     6481 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     6541 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     6601 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     6661 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6721 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     6781 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6841 aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt gggaagggcg
     6901 atcggtgcgg gcctcttcgc tattacgcca g
//
