LOCUS       sequence-242032-        6468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate acceptor vector for the assembly of EGFP-based mammalian
            recombinase reporters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6468)
  AUTHORS   Bessen JL, Afeyan LK, Dancik V, Koblan LW, Thompson DB, Leichner C, 
            Clemons PA, Liu DR
  TITLE     High-resolution specificity profiling and off-target prediction for 
            site-specific DNA recombinases.
  JOURNAL   Nat Commun. 2019 Apr 26;10(1):1937. doi: 10.1038/s41467-019-09987-0.
  PUBMED    31028261
REFERENCE   2  (bases 1 to 6468)
  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: "Nat 
            Commun."; date: "2019-04-26"; volume: "10(1):1937. doi"; pages: " 
            10.1038/s41467-019-09987-0"
COMMENT     Sequence Label: pCALNL-BsaI-EGFP
FEATURES             Location/Qualifiers
     source          1..6468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     terminator      complement(116..143)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(159..230)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(248..925)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             932..943
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    961..979
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    986..1004
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      1094..1103
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1100..1102,1103..1105,1106..1819)
                     /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(1100..1102,1103..1105,1106..1816)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1145..1166)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1406..1425)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1753..1774
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1906..1925)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1971..2026
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2025..2044)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2387..2403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2387..2403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2400..2422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2411..2427
                     /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(2435..2441,2442..2459,2460..2465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2480..2501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      2586..2720
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2740..2874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2777..2796)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2831..2850
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2942..2959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3113..3701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3193..3212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3872..4663,4664..4732))
                     /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     4495..4514
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4733..4837)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4868..5247
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          join(5466..6468,1..14)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     6406..6428
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
       61 aagaattgat taattcgagc gaacgcgtca ctaggagacc tagaagagct ataaacgcag
      121 aaaggcccac ccgaaggtga gccagtgtga ctctagtaga gagcgttcac cgacaaacaa
      181 cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggag
      241 atccttatta agcaccggtg gagtgacgac cttcagcacg ttcgtactgt tcaacgatgg
      301 tgtagtcttc gttgtgggag gtgatgtcca gtttgatgtc ggttttgtaa gcacccggca
      361 gctgaaccgg ttttttagcc atgtaggtgg ttttaacttc agcgtcgtag tgaccaccgt
      421 ctttcagttt cagacgcatt ttgatttcac ctttcagagc accgtcttcc gggtacatac
      481 gttcggtgga agcttcccaa cccatggttt ttttctgcat aaccggaccg tcggacggga
      541 agttggtacc acgcagttta actttgtaga tgaactcacc gtcttgcagg gaggagtcct
      601 gggtaacggt aacaacacca ccgtcttcga agttcataac acgttcccat ttgaaacctt
      661 ccgggaagga cagtttcagg tagtccggga tgtcagccgg gtgtttaacg taagctttgg
      721 aaccgtactg gaactgcggg gacaggatgt cccaagcgaa cggcagcgga ccacctttgg
      781 taactttcag tttagcggtc tgggtacctt cgtacggacg accttcacct tcaccttcga
      841 tttcgaactc gtgaccgtta acggaacctt ccatacgaac tttgaaacgc atgaactctt
      901 tgataacgtc ttcgctactc gccatagatc ctttctcctc tttcagatcc gtgctcagta
      961 tctctatcac tgatagggat gtcaatctct atcactgata gggagctctt ctggtctcct
     1021 tactcgagtc gctcggtacg atttaaattg aattctgcag tcgacggtac cgcgggcccg
     1081 ggatccaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1141 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1201 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1261 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1321 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1381 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1441 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1501 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1561 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1621 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1681 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1741 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1801 ggacgagctg tacaagtaaa gcggccgcac tcctcaggtg caggctgcct atcagaaggt
     1861 ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt ttccctctgc
     1921 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     1981 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata
     2041 tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata
     2101 tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa
     2161 acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt
     2221 ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt
     2281 tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct
     2341 cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc
     2401 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     2461 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2521 ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt agtcagcaac
     2581 catagtcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2641 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     2701 aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat
     2761 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     2821 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc
     2881 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     2941 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3001 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3061 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3121 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3181 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3241 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3301 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3361 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3421 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3481 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3541 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3601 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3661 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3721 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3781 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3841 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3901 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3961 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4021 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4081 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4141 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4201 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4261 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4321 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4381 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4441 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4501 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4561 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4621 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4681 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4741 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4801 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4861 ctgggtcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     4921 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     4981 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     5041 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     5101 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     5161 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     5221 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
     5281 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
     5341 gcagcgatgg gggcgggggg gggggtgcgg cggcagccaa tcagagcggc gcgctccgaa
     5401 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
     5461 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
     5521 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
     5581 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
     5641 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
     5701 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
     5761 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
     5821 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
     5881 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
     5941 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
     6001 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
     6061 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
     6121 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
     6181 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
     6241 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
     6301 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
     6361 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     6421 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttct
//
