LOCUS       sequence-11414-e        6216 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6216)
  AUTHORS   Hsiao EC, Nguyen TD, Ng JK, Scott MJ, Chang WC, Zahed H, Conklin BR
  TITLE     Constitutive Gs activation using a single-construct 
            tetracycline-inducible expression system in embryonic stem cells and
            mice.
  JOURNAL   Stem Cell Res Ther. 2011 Mar 4. 2(2):11.
  PUBMED    21375737
REFERENCE   2  (bases 1 to 6216)
  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: "Stem Cell 
            Res Ther. 2011 Mar 4. 2(2):11."
COMMENT     Sequence Label: pEnt R3L2 TetO(sh) mCh-Rs1-2
FEATURES             Location/Qualifiers
     source          1..6216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(63..651)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(143..162)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(741..1550)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1464..1483
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1542..1561
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1673..1772
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    1794..1827
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2513..2531
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2555..2573
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2597..2615
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2639..2657
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        2690..2728
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2725..2749
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2862..3434
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      3507..3516
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3513..4220
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3658..3676)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3926..3945
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4245..4301
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     polyA_signal    5665..5713
                     /note="synthetic polyadenylation signal"
     protein_bind    5740..5863
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5878..5901)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(5909..5936)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6028..6114)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
       61 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      121 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      181 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      241 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      301 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      361 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      421 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      481 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      541 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      601 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      661 ttgatctttt ctacggggtc tgacgctcag tggaacgggg cccaatctga ataatgttac
      721 aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
      781 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
      841 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
      901 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
      961 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     1021 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     1081 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     1141 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     1201 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgtttttcc ggggatcgca
     1261 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     1321 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     1381 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa gcgatagatt
     1441 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     1501 atgttggaat ttaatcgcgg cctcgacgtt tcccgttgaa tatggctcat aacacccctt
     1561 gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt
     1621 gcaatgtaac atcagagatt ttgagacacg ggccagagct gcagctggat ggcaaataat
     1681 gattttattt tgactgatag tgacctgttc gttgcaacaa attgataagc aatgctttct
     1741 tataatgcca actttgtaca agaaagctgg gtctagatat ctcgagttta aacataactt
     1801 cgtataatgt atgctatacg aagttatcat atgtggatcc ccgggtaccg agttggcgcg
     1861 cctgggagct cacggggaca gccccccccc aaagccccca gggatgtaat tacgtccctc
     1921 ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg
     1981 catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg
     2041 ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg
     2101 aaaaagcttt aggctgaaag agagatttag aatgacaggc gcgcctggga gctcacgggg
     2161 acagccccct cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc
     2221 agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag
     2281 cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt
     2341 ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaaagc tttaggctga
     2401 aagagagatt tagaatgaca ggcgcgccaa ctcgaattca ttgcagactt tcactggtac
     2461 cctgtttcct gaggttaatt aaggcccttt cgtcttcact cgagtttacc actccctatc
     2521 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga
     2581 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc
     2641 cctatcagtg atagagaaaa gtgaaagtcg agctcggtac ccgggtcgag gtaggcgtgt
     2701 acggtgggag gcctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg
     2761 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccc
     2821 cgaattcgag ctcggtaccc ggggatcctg agaacttcag ggtgagtttg gggacccttg
     2881 attgttcttt ctttttcgct attgtaaaat tcatgttata tggagggggc aaagttttca
     2941 gggtgttgtt tagaatggga agatgtccct tgtatcacca tggaccctca tgataatttt
     3001 gtttctttca ctttctactc tgttgacaac cattgtctcc tcttattttc ttttcatttt
     3061 ctgtaacttt ttcgttaaac tttagcttgc atttgtaacg aatttttaaa ttcacttttg
     3121 tttatttgtc agattgtaag tactttctct aatcactttt ttttcaaggc aatcagggta
     3181 tattatattg tacttcagca cagttttaga gaacaattgt tataattaaa tgataaggta
     3241 gaatatttct gcatataaat tctggctggc gtggaaatat tcttattggt agaaacaact
     3301 acaccctggt catcatcctg cctttctctt tatggttaca atgatataca ctgtttgaga
     3361 tgaggataaa atactctgag tccaaaccgg gcccctctgc taaccatgtt catgccttct
     3421 tctctttcct acagctcctg ggcaacgtgc tggttgttgt gctgtctcat cattttggca
     3481 aagaattcgt cgactgccta ggcgccgcca ccatggtgag caagggcgag gaggataaca
     3541 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     3601 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     3661 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     3721 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     3781 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     3841 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     3901 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     3961 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     4021 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     4081 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     4141 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     4201 gcatggacga gctgtacaag acctggtttc tcgagggcag cggcgcgacc aactttagcc
     4261 tgctgaaaca ggcgggcgat gtggaagaaa acccaggacc gaccggtatc aagacgatca
     4321 tcgccctgag ctacatcttc tgcctggtat tcgccgacta caaggacgat gatgacgcca
     4381 gcggccgcgc ccgggcggat cccccgggct gcaggatgga caaacttgat gctaatgtga
     4441 gttctgagga gggtttcggg tcagtggaga aggtggtgct gctcacgttt ctctcgacgg
     4501 ttatcctgat ggccatcttg gggaacctgc tggtgatggt ggctgtgtgc tgggacaggc
     4561 agctcaggaa aataaaaaca aattatttca ttgtatctct tgcttttgcg gatctgctgg
     4621 tttcggtgct ggtgatgccc tttggtgcca ttgagctggt tcaagacatc tggatttatg
     4681 gggaggtgtt ttgtcttgtt cggacatctc tggccgtcct gctcacaacg gcatcgattt
     4741 ttcacctgtg ctgcatttct ctggataggt attacgccat ctgctgccag cctttggtct
     4801 ataggaacaa gatgacccct ctgcgcatcg cattaatgct gggaggctgc tgggtcatcc
     4861 ccacgtttat ttcttttctc cctataatgc aaggctggaa taacattggc ataattgatt
     4921 tgatagaaaa gaggaagttc aaccagaact ctaactctac gtactgtgtc ttcatggtca
     4981 acaagcccta cgccatcacc tgctctgtgg tggccttcta catcccattt ctcctcatgg
     5041 tgctggccta ttaccgcatc tatgtcacag ctaaggagca tgcccatcag atccagatgt
     5101 tacaacgggc aggagcctcc tccgagagca ggcctcagtc ggcagaccag catagcactc
     5161 atcgcatgag gacagagacc aaagcagcca agaccctatg catcatcatg ggttgcttct
     5221 gcctctgctg ggcaccattc tttgtcacca atattgtgga tcctttcata gactacactg
     5281 tccctgggca ggtgtggact gctttcctct ggctcggcta tatcaattcc gggttgaacc
     5341 cttttctcta cgccttcttg aataagtctt ttagacgtgc cttcctcatc atcctctgct
     5401 gtgatgatga gcgctaccga agaccttcca ttctgggcca gactgtccct tgttcaacca
     5461 caaccattaa tggatccaca catgtactaa gggatgcagt ggagtgtggt ggccagtggg
     5521 agagtcagtg tcacccgcca gcaacttctc ctttggtggc tgctcagccc agtgacactt
     5581 agactagttc cggacctgca ggcgggactc tggggttcga aatgaccgac caagcgacgc
     5641 ccaacctgcc atcacgatgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt
     5701 ggttttttgt gtgaagcggc cgcattcaga gatcttaagc aactatgtat aataaagttg
     5761 aacgagaaac gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac
     5821 agactacata atactgtaaa acacaacata tccagtcact atggagatct taagtaacta
     5881 actaacagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
     5941 agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
     6001 cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
     6061 acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct
     6121 ggcagttccc tactctcgct tagtagttag acgtccccga gatccatgct agcggtaata
     6181 cggttatcca cagaatcagg ggataacgca ggaaag
//
