LOCUS       sequence-313442-        7636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-compatible destination vector for mammalian expression of 
            N-terminal EBFP protein under CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7636)
  TITLE     Gateway-compatible destination vector for mammalian expression of 
            N-terminal EBFP protein under CMV promoter
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7636)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pMH-EBFP
FEATURES             Location/Qualifiers
     source          1..7636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    50..174
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(199..204,205..222,223..229)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             283..963
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(349..368)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1283..1588
                     /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     1467..1486
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1629..1752)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1936..1953)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(2015..2035)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2020..2349
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2200..2335
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2359..2958
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2359..2378)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2855..2875
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3179..3648
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3778..3795)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3949..4537)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4029..4048)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4697..5488,5489..5557))
                     /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     5320..5339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5558..5662)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5693..6162
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     enhancer        6339..6642
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6643..6846
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6797..6817
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      6885..6894
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6891..6893,6894..6896,6897..7607)
                     /codon_start=1
                     /product="enhanced blue variant of GFP"
                     /label=EBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6936..6957)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7197..7216)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7544..7565
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 cgcgcctgaa ttctgcagtc gacggtaccg cgggcccatc gctcgagaga caagtttgta
       61 caaaaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
      121 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc
      181 gcattaggca ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg
      241 agttaggatc cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
      301 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag
      361 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag
      421 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg
      481 atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat
      541 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
      601 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt
      661 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca
      721 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc
      781 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg
      841 ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat
      901 gaattacaac agtactgcga tgagtggcag gcggggcgta atctagagga tccggcttac
      961 taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata
     1021 ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc gtattacagt
     1081 gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt
     1141 ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg
     1201 gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaccgg ctcttttgct
     1261 gacgagaaca ggggctggtg aaatgcagtt taaggtttac acctataaaa gagagagccg
     1321 ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt
     1381 gatccccctg gccagtgcac gtctactgtc agataaagtc ccccgtgaac tttacccggt
     1441 ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt
     1501 ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc
     1561 cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc
     1621 aggtcgacca tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat
     1681 gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcagctttct
     1741 tgtacaaagt ggctcgagca gggatccgcc cctctccctc ccccccccct aacgttactg
     1801 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
     1861 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
     1921 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     1981 cgtaggaatt cgccagcaca gtggtcgacc ctgtggaatg tgtgtcagtt agggtgtgga
     2041 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2101 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2161 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2221 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2281 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2341 ttttgcaaaa agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     2401 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     2461 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     2521 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     2581 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     2641 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     2701 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     2761 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     2821 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     2881 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     2941 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca
     3001 cgaccccatg catcgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
     3061 aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg
     3121 aaaaatacat aactgagaat agagaagttc agatcaaggt caggaacaga tggaacagct
     3181 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
     3241 acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     3301 ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag
     3361 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
     3421 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
     3481 aagagcccac aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta
     3541 cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg
     3601 ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcaca tgcagcatgt
     3661 atcaaaatta atttggtttt ttttcttaag tatttacatt aaatggccat agttgcatta
     3721 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3781 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3841 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3901 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3961 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4021 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4081 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4141 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4201 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4261 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4321 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4381 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4441 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4501 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4561 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4621 aaaaaggatc ttcacctaga tccttttgcg gccgcaaatc aatctaaagt atatatgagt
     4681 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4741 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4801 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4861 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4921 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4981 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5041 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5101 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5161 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5221 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5281 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5341 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5401 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5461 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5521 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5581 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5641 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgca gcctgaatat
     5701 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
     5761 ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
     5821 gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat
     5881 cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc
     5941 aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc
     6001 ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg
     6061 tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg
     6121 tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg ctcgtccggg
     6181 atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggcaagc tggccagcaa
     6241 cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc ctgcgtcggt
     6301 actagttcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     6361 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     6421 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     6481 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     6541 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     6601 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     6661 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     6721 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     6781 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     6841 agagctggtt tagtgaaccg tcagatccgc tagcgctacc ggtcgccacc atggtgagca
     6901 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     6961 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     7021 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     7081 ccctgaccca cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     7141 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     7201 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     7261 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     7321 acaacttcaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     7381 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     7441 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     7501 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     7561 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc ggactcagat
     7621 ctcgagctca agctgg
//
