LOCUS       sequence-168098-        4823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning plasmid derived from loxP reporter. The Kan and pA where 
            digested and removed with XhoI/MluI and replaced with two Esp3I 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4823)
  AUTHORS   Chaikind B, Bessen JL, Thompson DB, Hu JH, Liu DR
  TITLE     A programmable Cas9-serine recombinase fusion protein that operates 
            on DNA sequences in mammalian cells.
  JOURNAL   Nucleic Acids Res. 2016 Aug 11. pii: gkw707.
  PUBMED    27515511
REFERENCE   2  (bases 1 to 4823)
  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: "Nucleic 
            Acids Res. 2016 Aug 11. pii: gkw707."
COMMENT     Sequence Label: pCALNL-eGFP-Esp3I
FEATURES             Location/Qualifiers
     source          1..4823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        113..492
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        523..627
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(628..696,697..1488)
                     /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(846..865)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1659..2247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2148..2167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2401..2418
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    2486..2620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2510..2529)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2564..2583
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2640..2774
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    2859..2880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2895..2900,2901..2918,2919..2925)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2933..2949
                     /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     2938..2960
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2957..2973
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2957..2973
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3316..3335
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    3334..3389
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     3435..3454
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(join(3541..4254,4255..4257,4258..4260))
                     /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(3544..4254,4255..4257,4258..4260))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3586..3607)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3935..3954
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4194..4215
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4257..4266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4407..4426)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(4488..4510)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 cccccgcccc catcgctgca caaaataatt aaaaaataaa taaatacaaa attgggggtg
       61 gggagggggg ggagatgggg agagtgaagc agaacgtggg gctcacctcg accatggtaa
      121 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact
      181 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
      241 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
      301 gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
      361 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
      421 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca
      481 ataatcaatg tcgacccagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
      541 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
      601 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
      661 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
      721 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
      781 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
      841 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
      901 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
      961 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1021 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1081 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1141 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     1201 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     1261 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     1321 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     1381 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     1441 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     1501 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     1561 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1621 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1681 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1741 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1801 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1861 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1921 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1981 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2041 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2101 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2161 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2221 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2281 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2341 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2401 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     2461 cgttggccga ttcattaatg cagcggatcc agacatgata agatacattg atgagtttgg
     2521 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     2581 tgctttattt gtaaccatta taagctgcaa taaacaagtt agctttttgc aaaagcctag
     2641 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc
     2701 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     2761 agttaggggc gggactatgg ttgctgacta attgagatgc ggatccgctg gcacgacagg
     2821 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     2881 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     2941 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct tgggctgcag
     3001 gtcgagggat ctccataaga gaagagggac agctatgact gggagtagtc aggagaggag
     3061 gaaaaatctg gctagtaaaa catgtaagga aaattttagg gatgttaaag aaaaaaataa
     3121 cacaaaacaa aatataaaaa aaatctaacc tcaagtcaag gcttttctat ggaataagga
     3181 atggacagca gggggctgtt tcatatactg atgacctctt tatagccaac ctttgttcat
     3241 ggcagccagc atatgggcat atgttgccaa actctaaacc aaatactcat tctgatgttt
     3301 taaatgattt gccctcccat atgtccttcc gagtgagaga cacaaaaaat tccaacacac
     3361 tattgcaatg aaaataaatt tcctttatta gccagaagtc agatgctcaa ggggcttcat
     3421 gatgtcccca taatttttgg cagagggaaa aagatctcag tggtatttgt gagccagggc
     3481 attggccaca ccagccacca ccttctgata ggcagcctgc acctgaggag tgcggccgct
     3541 ttacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag
     3601 gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta
     3661 gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc
     3721 ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc
     3781 gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt
     3841 gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac
     3901 cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa
     3961 gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg
     4021 cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag
     4081 ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc
     4141 gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc
     4201 gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat
     4261 ggtggcgacc ggtggatccc gggcccgcgg taccgtcgac tgcagaattc aatttaaatc
     4321 gtaccgagcg actcgagcga gacggatggc agcgtctcaa cgcgttcgct cgaattaatc
     4381 aattctttgc caaaatgatg agacagcaca ataaccagca cgttgcccag gagctgtagg
     4441 aaaaagaaga aggcatgaac atggttagca gaggctctag agccgccggt cacacgccag
     4501 aagccgaacc ccgccctgcc ccgtcccccc cgaaggcagc cgtccccccg cggacagccc
     4561 cgaggctgga gagggagaag gggacggcgg cgcggcgacg cacgaaggcc ctccccgccc
     4621 atttccttcc tgccggcgcc gcaccgcttc gccccgcgcc cgctagaggg ggtgcggcgg
     4681 cgcctcccag atttcggctc cgcacagatt tgggacaaag gaagtccctg cgccctctcg
     4741 cacgattacc ataaaaggca atggctgcgg ctcgccgcgc ctcgacagcc gccggcgctc
     4801 cgggggccgc cgcgcccctc ccc
//
