LOCUS       sequence-250757-        5460 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 phiC31 attachment sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5460)
  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 5460)
  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)phiC31
FEATURES             Location/Qualifiers
     source          1..5460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    133..154
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(169..174,175..192,193..199)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    207..223
                     /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     212..234
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     231..247
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     231..247
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        268..286
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        329..1509
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          559..1501
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1458..1478
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1516..1549
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             complement(join(1556..2269,2270..2272,2273..2275))
                     /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(1559..2269,2270..2272,2273..2275))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1601..1622)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1950..1969
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2209..2230
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2423..2442)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2488..2543
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2542..2561)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2915..2934)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2916..2934)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2941..2958)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2941..2957)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2950..2972)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3099..3554)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3236..3257)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3448..3467
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3581..3685
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3686..3754,3755..4546)
                     /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(3904..3923)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4717..5305
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5206..5225
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(5459..5460,1..16)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
       61 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
      121 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
      181 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      241 ctatgaccat gattacgcca agctcgaaat taaccctcac taaagggaac aaaagctgga
      301 gctccaccgc ggtggcggcc gctctagagc tccggtgccc gtcagtgggc agagcgcaca
      361 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
      421 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
      481 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
      541 tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg
      601 ggttatggcc cttgcgtgcc ttgaattact tccacgcccc tggctgcagt acgtgattct
      661 tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc
      721 cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat
      781 ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt
      841 ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga
      901 tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca
      961 gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta
     1021 gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc
     1081 ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc
     1141 cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga
     1201 gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac
     1261 ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc
     1321 tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac
     1381 tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt
     1441 tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca
     1501 ggtgtcgtgg aattcgtgcc agggcgtgcc cttgggctcc ccgggcgcgc tcgagttact
     1561 tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca
     1621 tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt
     1681 tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga
     1741 gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct
     1801 tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc
     1861 ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg
     1921 tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga
     1981 tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca
     2041 tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc acgagggtgg
     2101 gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg
     2161 tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca
     2221 gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc accatgggcc
     2281 cccaactgag agaactcaaa ggttacccca gttgggggga tccgtcgacc gctagcagcg
     2341 gccgcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     2401 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     2461 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     2521 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     2581 atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa
     2641 caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     2701 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     2761 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     2821 tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag
     2881 cccaagcttg gcgtaatcat gggtacccaa ttcgccctat agtgagtcgt attacaattc
     2941 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     3001 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     3061 cccttcccaa cagttgcgca gcctgaatgg cgaatggcaa attgtaagcg ttaatatttt
     3121 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     3181 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     3241 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     3301 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     3361 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3421 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     3481 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3541 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3601 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3661 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3721 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3781 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3841 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3901 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     3961 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     4021 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     4081 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     4141 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     4201 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     4261 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     4321 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     4381 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     4441 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     4501 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     4561 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4621 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4681 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4741 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4801 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4861 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4921 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4981 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5041 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5101 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5161 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5221 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5281 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5341 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5401 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
//
