LOCUS       sequence-177135-        4889 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  pCAG promoter driving LSS-mOrange.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4889)
  AUTHORS   Weinberg BH, Pham NT, Caraballo LD, Lozanoski T, Engel A, Bhatia S, 
            Wong WW
  TITLE     Large-scale design of robust genetic circuits with multiple inputs 
            and outputs for mammalian cells.
  JOURNAL   Nat Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805.
  PUBMED    28346402
REFERENCE   2  (bases 1 to 4889)
  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 
            Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805."
COMMENT     Sequence Label: pCAG-LSS-mOrange
FEATURES             Location/Qualifiers
     source          1..4889
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     466..488
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     550..569
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      626..635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             632..1342
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     1045..1064
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1428..1447)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1493..1548
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1547..1566)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(1909..1925)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1909..1925)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1922..1944)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1933..1949
                     /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(1957..1963,1964..1981,1982..1987))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2002..2023
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      2108..2242
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2262..2396
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2299..2318)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2353..2372
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2464..2481)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2635..3223)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2715..2734)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3394..4185,4186..4254))
                     /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     4017..4036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4255..4359)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4390..4769
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
       61 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
      121 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
      181 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
      241 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
      301 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
      361 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
      421 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
      481 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
      541 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattgatta
      601 attcgagcga acgcgtaccg gtgccgccac catggtgagc aagggcgagg agaataacat
      661 ggccatcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg tgaacggcca
      721 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggctttc agaccgttaa
      781 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcttgt cccctcagtt
      841 cacctacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acctcaagct
      901 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
      961 gaccgtgact caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     1021 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gcatggaggc
     1081 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgaggaca agctcaggct
     1141 gaagctgaag gacggcggcc actacacctc cgaggtcaag accacctaca aggccaagaa
     1201 gcccgtgcag ttgcccggcg cctacatcgt cgacatcaag ttggacatca cctcccacaa
     1261 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     1321 catggacgag ctgtacaagt aagcggccgc actcctcagg tgcaggctgc ctatcagaag
     1381 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct
     1441 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     1501 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     1561 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     1621 tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg
     1681 aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt
     1741 ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat
     1801 gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt
     1861 ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt catagctgtt
     1921 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     1981 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2041 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca
     2101 accatagtcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2161 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     2221 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt
     2281 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2341 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct
     2401 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     2461 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2521 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2581 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2641 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2701 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2761 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2821 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2881 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2941 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3001 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3061 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3121 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3181 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3241 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3301 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3361 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3421 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3481 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3541 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3601 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3661 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3721 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3781 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3841 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3901 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3961 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4021 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4081 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4141 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4201 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4261 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4321 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4381 acctgggtcg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     4441 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     4501 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     4561 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     4621 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     4681 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     4741 tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc
     4801 tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt
     4861 gtgcagcgat gggggcgggg ggggggggg
//
