LOCUS       sequence-319502-        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  JOURNAL   Unpublished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            "Unpublished"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"
     LTR             1..470
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     enhancer        647..950
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        951..1154
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1105..1125
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1193..1202
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1199..1201,1202..1204,1205..1915)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1244..1265)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    1994..2118
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2143..2148,2149..2166,2167..2173)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2227..2907
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(2293..2312)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3227..3532
                     /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     3411..3430
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3573..3696)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3880..3897)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(3959..3979)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3964..4293
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4144..4279
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4303..4902
                     /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(4303..4322)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4799..4819
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5123..5592
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5722..5739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5893..6481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5973..5992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6641..7432,7433..7501))
                     /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     7264..7283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7502..7606)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
       61 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
      121 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
      181 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
      241 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
      301 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
      361 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
      421 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
      481 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggcaagctg
      541 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgactgatt ttatgcgcct
      601 gcgtcggtac tagttcatta gttcatagcc catatatgga gttccgcgtt acataactta
      661 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      721 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      781 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      841 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      901 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      961 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     1021 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     1081 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     1141 atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg tcgccaccat
     1201 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1261 cgacgtaaac ggccacaagt tcagcgtgag gggcgagggc gagggcgatg ccaccaacgg
     1321 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1381 cgtgaccacc ctgagccacg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca
     1441 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1501 caaggacgac ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1561 gaaccgcatc gagctgaagg gcgtcgactt caaggaggac ggcaacatcc tggggcacaa
     1621 gctggagtac aacttcaaca gccacaacat ctatatcatg gccgtcaagc agaagaacgg
     1681 catcaaggtg aacttcaaga tccgccacaa cgtggaggac ggcagcgtgc agctcgccga
     1741 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acagccacta
     1801 cctgagcacc cagtccgtgc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1861 gctggagttc cgcaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg
     1921 actcagatct cgagctcaag ctggcgcgcc tgaattctgc agtcgacggt accgcgggcc
     1981 catcgctcga gagacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa
     2041 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
     2101 acatatccag tcactatggc ggccgcatta ggcaccccag gctttacact ttatgcttcc
     2161 ggctcgtata atgtgtggat tttgagttag gatccgtcga gattttcagg agctaaggaa
     2221 gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt
     2281 aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag
     2341 ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc
     2401 tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa
     2461 gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa
     2521 actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac
     2581 atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt
     2641 attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta
     2701 aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg
     2761 caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc
     2821 ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg gcaggcgggg
     2881 cgtaatctag aggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg
     2941 atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta
     3001 tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc
     3061 atatatgatg tcaatatctc cggtctggta agcacaacca tgcagaatga agcccgtcgt
     3121 ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt
     3181 attgaaatga ccggctcttt tgctgacgag aacaggggct ggtgaaatgc agtttaaggt
     3241 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat
     3301 tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctac tgtcagataa
     3361 agtcccccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac
     3421 caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca
     3481 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg
     3541 ctcccttata cacagccagt ctgcaggtcg accatagtga ctggatatgt tgtgttttac
     3601 agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat
     3661 tttacgtttc tcgttcagct ttcttgtaca aagtggctcg agcagggatc cgcccctctc
     3721 cctccccccc ccntaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     3781 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     3841 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     3901 gtctgttgaa tgtcgtgaag gaagcgtagg aattcgccag cacagtggtc gaccctgtgg
     3961 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4021 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4081 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4141 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4201 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4261 ggaggctttt ttggaggcct aggcttttgc aaaaagctta ccatgaccga gtacaagccc
     4321 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     4381 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     4441 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     4501 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     4561 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     4621 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     4681 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     4741 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     4801 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     4861 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg
     4921 cagcgcccga ccgaaaggag cgcacgaccc catgcatcga taaaataaaa gattttattt
     4981 agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa
     5041 gtaacgccat tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca
     5101 aggtcaggaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
     5161 tcctgccccg gctcagggcc aagaacagat ggaacagctg aatatgggcc aaacaggata
     5221 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg
     5281 tccagccctc agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg
     5341 aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc
     5401 gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct
     5461 ccgattgact gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc
     5521 gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc
     5581 gggggtcttt cacatgcagc atgtatcaaa attaatttgg ttttttttct taagtattta
     5641 cattaaatgg ccatagttgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5701 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5761 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5821 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5881 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5941 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6001 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6061 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6121 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6181 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6241 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6301 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6361 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6421 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6481 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6541 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt tgcggccgca
     6601 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6661 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6721 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6781 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6841 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6901 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6961 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7021 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7081 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7141 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7201 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7261 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7321 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7381 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7441 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7501 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7561 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7621 aagtgccacc tgcagc
//
