LOCUS       sequence-384843-        8163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1 Integratable mCherry Reporter for synZiFTR ZF4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8163)
  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 8163)
  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-012
FEATURES             Location/Qualifiers
     source          1..8163
                     /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     1092..1116
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1139..1148
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1145..1855
                     /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(1290..1308)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1558..1577
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1941..1960)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2006..2061
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2060..2079)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    2407..2631
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2696..3195
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2731..2754)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3128..3147
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3219..3815
                     /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(3219..3238)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3715..3735
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3825..3881
                     /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             3882..4583
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4701..4822
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    4963..5464
                     /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(5666..5682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5666..5682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5679..5701)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5690..5706
                     /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(5714..5720,5721..5738,5739..5744))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5759..5780
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5839..6034
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5885..6020
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6040..6174
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6242..6259)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6413..7001)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6493..6512)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7172..7963,7964..8032))
                     /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     7795..7814
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8033..8137)
                     /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 aatcgtcgac
      841 cgggtttcgt aacaatcgca tgaggattcg caacgccttg gacgacgccg atgtagaagt
      901 cccgtctcag taaaggtgga cgacgccgat gtagaagaat cggactgcct tcgtaggacg
      961 acgccgatgt agaaggtatc agtcgcctcg gaaggacgac gccgatgtag aagattcgta
     1021 agaggctcac tctcccttac acggagtgga taactagtta ggcgtgtacg gtgggaggcc
     1081 tatataagca gagctcgttt agtgaaccgt cagatcgcct ggaacgcgta ccggtgtcgc
     1141 caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     1201 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     1261 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     1321 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     1381 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     1441 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     1501 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     1561 cgtaatgcag aagaagacca tgggctggca ggcctcctcc gagcggatgt accccgagga
     1621 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     1681 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     1741 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     1801 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagcggc
     1861 cgcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     1921 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     1981 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     2041 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     2101 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     2161 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     2221 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     2281 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     2341 tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag
     2401 ggtaccctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2461 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2521 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2581 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     2641 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc gtaccgggta
     2701 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact
     2761 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa
     2821 ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag
     2881 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc
     2941 actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc
     3001 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg
     3061 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga
     3121 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca
     3181 tctccgggcc tttcgatatc tcgcacagtg ccgccaccat gaccgagtac aagcccacgg
     3241 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     3301 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     3361 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     3421 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     3481 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     3541 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     3601 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     3661 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     3721 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     3781 cgcgcacctg gtgcatgacc cgcaagcccg gtgccggaag cggagctact aacttcagcc
     3841 tgctgaagca ggctggagac gtggaggaga accctggacc tatgagcgag ctgattaagg
     3901 agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca
     3961 catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg
     4021 agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca
     4081 agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg
     4141 gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg
     4201 acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca
     4261 catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc
     4321 tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg
     4381 ggagccatct gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc
     4441 tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca
     4501 acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca
     4561 aactggggca caagcttaat taatctagat aactgatcat aatcagccat accacatttg
     4621 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     4681 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     4741 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4801 ccaaactcat caatgtatct taggcgcgcc ctgtccctag tggccccact gtggggtgga
     4861 ggggacagat aaaagtaccc agaaccagag ccacattaac cggccctggg aatataaggt
     4921 ggtcccagct cggggacaca ggatccctgg aggcagcaaa catgctgtcc tgaagtggac
     4981 ataggggccc gggttggagg aagaagacta gctgagctct cggacccctg gaagatgcca
     5041 tgacaggggg ctggaagagc tagcacagac tagagaggta aggggggtag gggagctgcc
     5101 caaatgaaag gagtgagagg tgacccgaat ccacaggaga acggggtgtc caggcaaaga
     5161 aagcaagagg atggagaggt ggctaaagcc agggagacgg ggtactttgg ggttgtccag
     5221 aaaaacggtg atgatgcagg cctacaagaa ggggaggcgg gacgcaaggg agacatccgt
     5281 cggagaaggc catcctaaga aacgagagat ggcacaggcc ccagaaggag aaggaaaagg
     5341 gaacccagcg agtgaagacg gcatggggtt gggtgaggga ggagagatgc ccggagagga
     5401 cccagacacg gggaggatcc gctcagagga catcacgtgg tgcagcgccg agaaggaagt
     5461 gctccggaaa gagcatcctt gggcagcaac acagcagaga gcaaggggaa gagggagtgg
     5521 aggaagacgg aacctgaagg aggcggcagg gaaggatctg ggccagccgt agaggtgacc
     5581 caggccacaa gctgcagaca gaaagcggca caggcccagg ggagagaatg ccctcgacct
     5641 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5701 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5761 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5821 tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     5881 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     5941 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     6001 aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat
     6061 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     6121 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc
     6181 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     6241 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6301 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6361 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6421 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6481 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6541 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6601 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6661 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6721 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6781 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6841 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6901 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6961 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     7021 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     7081 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     7141 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7201 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7261 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7321 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7381 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7441 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7501 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7561 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7621 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7681 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7741 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7801 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7861 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7921 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7981 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     8041 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     8101 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8161 ctg
//
