LOCUS       sequence-244154-        4463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid has an inverted CaMV 35S promoter sequence (reverse 
            complement) flanked by attB and attP attachment sites of integrases 
            2, 4, and 5. EGFP coding sequence is in the forward orientation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4463)
  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 4463)
  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-p35S(rc)2_4_5-GFP
FEATURES             Location/Qualifiers
     source          1..4463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(636..980)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(672..692)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             join(1213..1215,1216..1218,1219..1932)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1258..1279)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1519..1538)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1866..1887
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      1951..2203
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2242..2258)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2242..2258)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2255..2277)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2266..2282
                     /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(2290..2296,2297..2314,2315..2320))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2335..2356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2473..2490)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2644..3232)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2724..2743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3403..4194,4195..4263))
                     /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     4026..4045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4264..4368)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4436..4454
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ggacggcgca
      421 gaaggggagt agctcttcgc cggaccgtcg acatactgct cagctcgtct tccaaagagc
      481 gcccaacgcg acctgaaatt tgaataagac tgctgcttgt gtaaaggcga tgattgagcg
      541 ccggatcagg gagtggacgg cctgggagcg ctacacgctg tggctgcggt cggtgcaatt
      601 ggattaggag tttggaagat aatttggcac agttagtcct ctccaaatga aatgaacttc
      661 cttatataga ggaagggtct tgcgaaggat agtgggattg tgcgtcatcc cttacgtcag
      721 tggagatatc acatcaatcc acttgctttg aagacgtggt tggaacgtct tctttttcca
      781 cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag gcatcttcaa
      841 cgatggcctt tcctttatcg caatgatggc atttgtagga gccaccttcc ttttccacta
      901 tcttcacaat aaagtgacag atagctgggc aatggaatcc gaggaggttt ccggatatta
      961 ccctttgttg aaaagtctca tatgaaatta gggtctatat agttataatt tgatgaatta
     1021 cagatcaggg tgcgcaagtt gtcaacgctc ccaggagagt tatcgacttg cgtattaggg
     1081 aaacgaatta caaattattt gaccgctaat tcaaatcaag ggttgaaaac tttgtaattt
     1141 ttgagccaag agcagtgcct tgcagaagtt ctcgggcgag aatctcgcgt agacacatac
     1201 atgagcggat ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1261 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1321 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1381 ccctggccca ccctcgtgac caccttcacc tacggcgtgc agtgcttcag ccgctacccc
     1441 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1501 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1561 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1621 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1681 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1741 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1801 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1861 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactcacgg catggacgag
     1921 ctgtacaagt aaagcggccg cccggctgca gatcgttcaa acatttggca ataaagtttc
     1981 ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac
     2041 gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg
     2101 attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac
     2161 taggataaat tatcgcgcgc ggtgtcatct atgttactag atccgatgat aagctgtcaa
     2221 acatgagaat tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2281 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     2341 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2401 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2461 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2521 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2581 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2641 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2701 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2761 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2821 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2881 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2941 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3001 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3061 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     3121 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3181 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3241 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3301 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3361 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3421 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3481 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3541 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3601 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3661 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3721 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3781 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3841 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3901 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3961 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4021 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4081 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4141 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4201 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4261 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4321 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4381 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     4441 gcgtatcacg aggccctttc gtc
//
