LOCUS       sequence-202924-        7159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sleeping Beaquty (SB) vector encoding G418 resistance and GFP from 
            two separate promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7159)
  AUTHORS   Chicaybam L, Barcelos C, Peixoto B, Carneiro M, Limia CG, Redondo P,
            Lira C, Paraguassu-Braga F, Vasconcelos ZF, Barros L, Bonamino MH
  TITLE     An Efficient Electroporation Protocol for the Genetic Modification 
            of Mammalian Cells.
  JOURNAL   Front Bioeng Biotechnol. 2017 Jan 23;4:99. doi: 
            10.3389/fbioe.2016.00099. eCollection 2016.
  PUBMED    28168187
REFERENCE   2  (bases 1 to 7159)
  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: "Front 
            Bioeng Biotechnol."; date: "2017-01-23"; volume: "4:99. doi"; pages:
            " 10.3389/fbioe.2016.00099. eCollection 2016"
COMMENT     Sequence Label: pT3-Neo-EF1a-GFP
FEATURES             Location/Qualifiers
     source          1..7159
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        64..1242
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          295..1233
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1190..1210
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1319..1328
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1325..1327,1328..1330,1331..2041)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1325..1327,1328..1330,1331..2041)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1370..1391)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1631..1650)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1978..1999
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2520..2529
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3355..3373)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3356..3373)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3393..3410)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3399..3623
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4062..4078)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4062..4078)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4075..4097)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4086..4102
                     /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(4110..4116,4117..4134,4135..4140))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4155..4176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4293..4310)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4464..5052)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4544..4563)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5223..6014,6015..6083))
                     /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     5846..5865
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6084..6188)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6256..6274
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6312..6334)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6434..6453
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6647..6669
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6662..6678
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7060..7080)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa aggagtggga
       61 attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
      121 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
      181 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
      241 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt
      301 gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga
      361 attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
      421 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
      481 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
      541 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
      601 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
      661 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
      721 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
      781 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
      841 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
      901 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
      961 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
     1021 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     1081 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     1141 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     1201 acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaattct gcagtcgatc
     1261 gacggtaccg cgggcccggg atccaggcct aagcttacgc gtcctagcgc taccggtcgc
     1321 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1381 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1441 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1501 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1561 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1621 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1681 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1741 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1801 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1861 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1921 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1981 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     2041 gtccggactc agatctcgac tagctagtag ctagctagct agtcgagctc aagcttcgat
     2101 actagtttaa ttaagatctc gagggaatga aagaccccac ctgtaggttt ggcaagctag
     2161 cttaagtaac gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag
     2221 atcaaggtta ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc
     2281 agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag
     2341 cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt
     2401 gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc
     2461 cgagctcaat aaaagagccc acaacccctc actcggcgcg ccagtccgga tccaccggtg
     2521 ccaccatggc cgaacaggat ggactgcacg caggctcccc cgccgcttgg gtggaaagac
     2581 tgtttggata cgactgggct cagcagacaa tcggatgctc tgacgcagcc gtgttccggc
     2641 tgagtgctca ggggcgcccc gtgctgtttg tcaagacaga cctgtcagga gcactgaacg
     2701 agctgcagga tgaagcagcc cgcctgagct ggctggctac cacaggagtg ccttgtgctg
     2761 cagtcctgga cgtggtcact gaggcaggcc gagattggct gctgctgggg gaagtgccag
     2821 gacaggacct gctgagctcc cacctggcac cagctgagaa ggtctccatc atggcagatg
     2881 ccatgcggag actgcatact ctggaccccg ccacctgccc tttcgatcac caggctaaac
     2941 atcgcattga gagggctcgc acccgaatgg aagcaggcct ggtggaccag gacgatctgg
     3001 atgaggaaca ccaggggctg gctcctgcag agctgtttgc acggctgaaa gccagaatgc
     3061 cagacggaga agatctggtg gtcacccatg gcgacgcttg cctgcccaac atcatggtgg
     3121 agaatggcag attcagtggg tttattgatt gtggacgact gggcgtcgcc gacagatacc
     3181 aggatatcgc cctggctaca agagacattg cagaggaact gggcggggaa tgggccgata
     3241 ggttcctggt gctgtatggc atcgccgcac ctgattcaca gaggattgct ttttacagac
     3301 tgctggatga gtttttctaa gcggccgctc gagcatgcat ctagagggcc ctattctata
     3361 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     3421 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3481 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3541 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3601 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctgca ttaatggatc
     3661 cccttgaaat acatccacag gtacacctcc aattgactca aatgatgtca attagtctat
     3721 cagaagcttc taaagccatg acatcatttt ctggaatttt ccaagctgtt taaaggcaca
     3781 gtcaacttag tgtatgtaaa cttctgaccc actggaattg tgatacagtg aattataagt
     3841 gaaataatct gtctgtaaac aattgttgga aaaatgactt gtgtcatgca caaagtagat
     3901 gtcctaactg acttgccaaa actattgttt gttaacaaga aatttgtgga gtagttgaaa
     3961 aacgagtttt aatgactcca acttaagtgt atgtaaactt ccgacttcaa ctgtataggt
     4021 ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4081 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4141 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4201 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4261 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4321 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4381 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4441 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4501 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4561 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4621 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4681 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4741 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4801 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4861 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4921 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4981 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5041 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5101 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5161 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5221 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5281 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5341 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5401 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5461 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5521 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5581 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5641 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5701 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5761 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5821 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5881 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5941 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6001 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6061 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6121 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6181 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     6241 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     6301 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     6361 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     6421 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     6481 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg
     6541 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     6601 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     6661 tgtaaaacga cggccagtga attcgagctc ggtaccctac agttgaagtc ggaagtttac
     6721 atacacttaa gttggagtca ttaaaactcg tttttcaact actccacaaa tttcttgtta
     6781 acaaacaata gttttggcaa gtcagttagg acatctactt tgtgcatgac acaagtcatt
     6841 tttccaacaa ttgtttacag acagattatt tcacttataa ttcactgtat cacaattcca
     6901 gtgggtcaga agtttacata cactaagttg actgtgcctt taaacagctt ggaaaattcc
     6961 agaaaatgat gtcatggctt tagaagcttc tgatagacta attgacatca tttgagtcaa
     7021 ttggaggtgt acctgtggat gtatttcaag gaattctgtg gaatgtgtgt cagttagggt
     7081 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat atcgataagc
     7141 tttgcaaaga tggataaag
//
