LOCUS       sequence-250843-        4409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid has a CaMV 35S 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 4409)
  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 4409)
  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-GFP(rc)Bxb1
FEATURES             Location/Qualifiers
     source          1..4409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(42..386)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(78..98)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     promoter        complement(445..463)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(484..500)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(484..500)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(497..519)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    508..524
                     /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(532..538,539..556,557..562))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    577..598
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(715..732)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(886..1474)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(966..985)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1645..2436,2437..2505))
                     /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     2268..2287
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2506..2610)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2637..3092
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2724..2743)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2934..2955
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3219..3241
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3233..3250
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3234..3250
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3257..3276
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3257..3275
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(3291..3544)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3569..3626
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attP site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     CDS             join(3630..3632,3633..3635,3636..4349)
                     /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             join(3630..3632,3633..3635,3636..4346)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3675..3696)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3936..3955)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4283..4304
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    4356..4405
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
ORIGIN
        1 tcaattggat taggagtttg gaagataatt tggcacagtt agtcctctcc aaatgaaatg
       61 aacttcctta tatagaggaa gggtcttgcg aaggatagtg ggattgtgcg tcatccctta
      121 cgtcagtgga gatatcacat caatccactt gctttgaaga cgtggttgga acgtcttctt
      181 tttccacgat gctcctcgtg ggtgggggtc catctttggg accactgtcg gcagaggcat
      241 cttcaacgat ggcctttcct ttatcgcaat gatggcattt gtaggagcca ccttcctttt
      301 ccactatctt cacaataaag tgacagatag ctgggcaatg gaatccgagg aggtttccgg
      361 atattaccct ttgttgaaaa gtctcatatg aaattagggt ctatatagtt ataatttgat
      421 gaattagagc tccagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc
      481 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
      541 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
      601 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
      661 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
      721 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
      781 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
      841 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
      901 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
      961 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     1021 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     1081 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     1141 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     1201 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     1261 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     1321 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     1381 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     1441 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     1501 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     1561 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     1621 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     1681 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     1741 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     1801 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     1861 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     1921 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     1981 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2041 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2101 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     2161 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     2221 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     2281 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     2341 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     2401 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     2461 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     2521 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2581 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc
     2641 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2701 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2761 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2821 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2881 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2941 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3001 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3061 gaattttaac aaaatattaa cgcttacaat ttgccattcg ccattcaggc tgcgcaactg
     3121 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     3181 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     3241 gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtaccc gatctagtcc
     3301 tcatagatga caccgcgcgc gataatttat ccaagtttgc gcgcgatatt ttgttttcta
     3361 tcgcgtatta aatgtataat tgcgggactc taatcataaa aacccatctc ataaataacg
     3421 tcatgcatta catgttaaat attacatgct taacgtaatt caacagaaat tatatgataa
     3481 tcatcgcaag accggcaaca ggattcaatc ttaagaaact ttattgccaa atgtttgaac
     3541 gatcggggaa attcgcggcc gcaagcttgt cgtggtttgt ctggtcaacc accgcggtct
     3601 cagtggtgta cggtacaaac cccgacccca tggtgagcaa gggcgaggag ctgttcaccg
     3661 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     3721 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     3781 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     3841 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3901 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     3961 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     4021 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     4081 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     4141 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     4201 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     4261 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     4321 ctctcggcat ggacgagctg tacaagtaac tcgaggcccg gatgatcctg acgacggaga
     4381 ccgccgtcgt cgacaagccg gccgagaat
//
