LOCUS       sequence-385000-        8164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1 Integratable mCherry Reporter for synZiFTR ZF5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8164)
  AUTHORS   Li HS, Israni DV, Gagnon KA, Gan KA, Raymond MH, Sander JD, Roybal 
            KT, Joung JK, Wong WW, Khalil AS
  TITLE     Multidimensional control of therapeutic human cell function with 
            synthetic gene circuits.
  JOURNAL   Science. 2022 Dec 16;378(6625):1227-1234. doi: 
            10.1126/science.ade0156. Epub 2022 Dec 15.
  PUBMED    36520914
REFERENCE   2  (bases 1 to 8164)
  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"; doi: 
            "10.1126/science.ade0156"; journalName: "Science"; date: 
            "2022-12-16- 16"; volume: "378"; issue: "6625"; pages: "1227-1234"
COMMENT     Sequence Label: pMN-013
FEATURES             Location/Qualifiers
     source          1..8164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    127..656
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     1093..1117
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1140..1149
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1146..1856
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWQASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1291..1309)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1559..1578
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1942..1961)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2007..2062
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2061..2080)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    2408..2632
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2697..3196
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2732..2755)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3129..3148
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3220..3816
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3220..3239)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3716..3736
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3826..3882
                     /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"
     CDS             3883..4584
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4702..4823
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    4964..5465
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(5667..5683)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5667..5683)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5680..5702)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5691..5707
                     /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(5715..5721,5722..5739,5740..5745))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5760..5781
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5840..6035
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5886..6021
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6041..6175
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6243..6260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6414..7002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6494..6513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7173..7964,7965..8033))
                     /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     7796..7815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8034..8138)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agtgaagggg caggagagcc aggggcatga gatggtggac
       61 gaggaagggg gacagggaag cctgagcgcc tctcctgggc ttgccaagga ctcaaaccca
      121 gaagcccaga gcagggcctt agggaagcgg gaccctgctc tgggcggagg aatatgtccc
      181 agatagcact ggggactctt taaggaaaga aggatggaga aagagaaagg gagtagaggc
      241 ggccacgacc tggtgaacac ctaggacgca ccattctcac aaagggagtt ttccacacgg
      301 acacccccct cctcaccaca gccctgccag gacggggctg gctactggcc ttatctcaca
      361 ggtaaaactg acgcacggag gaacaatata aattggggac tagaaaggtg aagagccaaa
      421 gttagaactc aggaccaact tattctgatt ttgtttttcc aaactgcttc tcctcttggg
      481 aagtgtaagg aagctgcagc accaggatca gtgaaacgca ccagacagcc gcgtcagagc
      541 agctcaggtt ctgggagagg gtagcgcagg gtggccactg agaaccgggc aggtcacgca
      601 tccccccctt ccctcccacc ccctgccaag ctctccctcc caggatcctc tctggctcca
      661 tcgtaagcaa accttagagg ttctggcaag gagagagatg gctccaggaa atgggggtgt
      721 gtcaccagat aaggaatctg cctaacagga ggtgggggtt agacccaata tcaggagact
      781 aggaaggagg aggcctaagg atggggcttt tctgtcacca atcgccggcg aattcgtcga
      841 ccgggtttcg taacaatcgc atgaggattc gcaacgcctt tgaagcagtc gacgccgaag
      901 tcccgtctca gtaaaggttg aagcagtcga cgccgaagaa tcggactgcc ttcgtatgaa
      961 gcagtcgacg ccgaaggtat cagtcgcctc ggaatgaagc agtcgacgcc gaagattcgt
     1021 aagaggctca ctctccctta cacggagtgg ataactagtt aggcgtgtac ggtgggaggc
     1081 ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggaacgcgt accggtgtcg
     1141 ccaccatggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     1201 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     1261 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     1321 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     1381 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     1441 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     1501 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     1561 ccgtaatgca gaagaagacc atgggctggc aggcctcctc cgagcggatg taccccgagg
     1621 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     1681 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     1741 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     1801 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaagcgg
     1861 ccgcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct
     1921 ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa
     1981 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     2041 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     2101 tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa
     2161 caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt
     2221 attccataga aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt
     2281 tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct
     2341 ctcctgacta ctcccagtca tagctgtccc tcttctctta tgaagatccc tcgacctgca
     2401 gggtaccctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2461 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2521 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2581 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2641 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cgtaccgggt
     2701 aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac
     2761 ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca
     2821 accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa
     2881 gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct
     2941 cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg
     3001 cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg
     3061 gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg
     3121 aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc
     3181 atctccgggc ctttcgatat ctcgcacagt gccgccacca tgaccgagta caagcccacg
     3241 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     3301 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     3361 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     3421 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     3481 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     3541 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     3601 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     3661 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     3721 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     3781 ccgcgcacct ggtgcatgac ccgcaagccc ggtgccggaa gcggagctac taacttcagc
     3841 ctgctgaagc aggctggaga cgtggaggag aaccctggac ctatgagcga gctgattaag
     3901 gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc
     3961 acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc
     4021 gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc
     4081 aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag
     4141 ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag
     4201 gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc
     4261 acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg
     4321 ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc
     4381 gggagccatc tgatcgcaaa catcaagacc acatatagat ccaagaaacc cgctaagaac
     4441 ctcaagatgc ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac
     4501 aacgagacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc
     4561 aaactggggc acaagcttaa ttaatctaga taactgatca taatcagcca taccacattt
     4621 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     4681 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     4741 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4801 tccaaactca tcaatgtatc ttaggcgcgc cctgtcccta gtggccccac tgtggggtgg
     4861 aggggacaga taaaagtacc cagaaccaga gccacattaa ccggccctgg gaatataagg
     4921 tggtcccagc tcggggacac aggatccctg gaggcagcaa acatgctgtc ctgaagtgga
     4981 cataggggcc cgggttggag gaagaagact agctgagctc tcggacccct ggaagatgcc
     5041 atgacagggg gctggaagag ctagcacaga ctagagaggt aaggggggta ggggagctgc
     5101 ccaaatgaaa ggagtgagag gtgacccgaa tccacaggag aacggggtgt ccaggcaaag
     5161 aaagcaagag gatggagagg tggctaaagc cagggagacg gggtactttg gggttgtcca
     5221 gaaaaacggt gatgatgcag gcctacaaga aggggaggcg ggacgcaagg gagacatccg
     5281 tcggagaagg ccatcctaag aaacgagaga tggcacaggc cccagaagga gaaggaaaag
     5341 ggaacccagc gagtgaagac ggcatggggt tgggtgaggg aggagagatg cccggagagg
     5401 acccagacac ggggaggatc cgctcagagg acatcacgtg gtgcagcgcc gagaaggaag
     5461 tgctccggaa agagcatcct tgggcagcaa cacagcagag agcaagggga agagggagtg
     5521 gaggaagacg gaacctgaag gaggcggcag ggaaggatct gggccagccg tagaggtgac
     5581 ccaggccaca agctgcagac agaaagcggc acaggcccag gggagagaat gccctcgacc
     5641 tgcagcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5701 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5761 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5821 ctgtcgtgcc agcggatccg catctcaatt agtcagcaac catagtcccg cccctaactc
     5881 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5941 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     6001 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta
     6061 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     6121 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg
     6181 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     6241 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     6301 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     6361 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6421 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6481 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6541 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6601 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6661 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6721 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6781 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6841 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6901 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6961 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     7021 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     7081 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     7141 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     7201 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     7261 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     7321 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7381 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7441 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7501 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7561 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7621 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7681 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7741 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7801 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7861 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7921 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7981 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     8041 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     8101 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     8161 cctg
//
