LOCUS       sequence-250763-        5495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid has an EF1 alpha promoter in a forward sequence orientation 
            and an inverted EGFP coding sequence (reverse complement) flanked by
            attB and attP integrase Bxb1 attachment sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5495)
  AUTHORS   Gomide MS, Sales TT, Barros LRC, Limia CG, de Oliveira MA, 
            Florentino LH, Barros LMG, Robledo ML, Jose GPC, Almeida MSM, Lima 
            RN, Rehen SK, Lacorte C, Melo EO, Murad AM, Bonamino MH, Coelho CM, 
            Rech E
  TITLE     Genetic switches designed for eukaryotic cells and controlled by 
            serine integrases.
  JOURNAL   Commun Biol. 2020 May 22;3(1):255. doi: 10.1038/s42003-020-0971-8.
  PUBMED    32444777
REFERENCE   2  (bases 1 to 5495)
  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: "Commun 
            Biol."; date: "2020-05-22"; volume: "3(1):255. doi"; pages: " 
            10.1038/s42003-020-0971-8"
COMMENT     Sequence Label: INCTbiosyn-pEF-GFP(rc)Bxb1
FEATURES             Location/Qualifiers
     source          1..5495
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    139..160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(175..180,181..198,199..205)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    213..229
                     /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)."
     primer_bind     218..240
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     237..253
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     237..253
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        274..292
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        335..1515
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          565..1507
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1464..1484
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1522..1571
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     CDS             complement(join(1578..2291,2292..2294,2295..2297))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1581..2291,2292..2294,2295..2297))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1623..1644)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1972..1991
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2231..2252
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2301..2358
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attP site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     primer_bind     complement(2464..2483)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2529..2584
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2583..2602)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2956..2975)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2957..2975)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2982..2999)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2982..2998)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2991..3013)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3140..3595)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3277..3298)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3489..3508
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3622..3726
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3727..3795,3796..4587)
                     /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     complement(3945..3964)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4758..5346
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5247..5266
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
       61 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
      121 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
      181 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
      241 aaacagctat gaccatgatt acgccaagct cgaaattaac cctcactaaa gggaacaaaa
      301 gctggagctc caccgcggtg gcggccgctc tagagctccg gtgcccgtca gtgggcagag
      361 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc
      421 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
      481 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
      541 aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc
      601 tttacgggtt atggcccttg cgtgccttga attacttcca cgcccctggc tgcagtacgt
      661 gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta
      721 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
      781 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
      841 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
      901 ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
      961 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
     1021 ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc
     1081 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
     1141 gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc
     1201 gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga
     1261 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac
     1321 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     1381 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     1441 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     1501 atttcaggtg tcgtggaatt ctcggccggc ttgtcgacga cggcggtctc cgtcgtcagg
     1561 atcatccggg cctcgagtta cttgtacagc tcgtccatgc cgagagtgat cccggcggcg
     1621 gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt gctcagggcg
     1681 gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg
     1741 ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg
     1801 aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt gtggctgttg
     1861 tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc
     1921 agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag
     1981 ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg catggcggac
     2041 ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg cacgccgtag
     2101 gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac
     2161 ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac gctgaacttg
     2221 tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc
     2281 tcgcccttgc tcaccatggg gtcggggttt gtaccgtaca ccactgagac cgcggtggtt
     2341 gaccagacaa accacgacgg atccgtcgac cgctagcagc ggccgcactc ctcaggtgca
     2401 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     2461 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     2521 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     2581 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     2641 ttagagtttg gcaacatatg ccatatgctg gctgccatga acaaaggtgg ctataaagag
     2701 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     2761 cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa
     2821 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     2881 tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt ggcgtaatca
     2941 tgggtaccca attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa
     3001 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     3061 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     3121 agcctgaatg gcgaatggca aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3181 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3241 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3301 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3361 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3421 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3481 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3541 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     3601 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3661 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3721 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3781 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3841 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3901 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3961 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     4021 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4081 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4141 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4201 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4261 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4321 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4381 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4441 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4501 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4561 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4621 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4681 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4741 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4801 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4861 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4921 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4981 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5041 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5101 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5161 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5221 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5281 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5341 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5401 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5461 tgagctgata ccgctcgccg cagccgaacg accga
//
