LOCUS       sequence-384841-        8152 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1 Integratable mCherry Reporter for synZiFTR ZF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8152)
  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 8152)
  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-009
FEATURES             Location/Qualifiers
     source          1..8152
                     /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..3184
                     /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     3117..3136
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3208..3804
                     /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(3208..3227)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3704..3724
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3814..3870
                     /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             3871..4572
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4690..4811
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    4952..5453
                     /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(5655..5671)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5655..5671)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5668..5690)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5679..5695
                     /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(5703..5709,5710..5727,5728..5733))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5748..5769
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5828..6023
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5874..6009
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6029..6163
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6231..6248)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6402..6990)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6482..6501)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7161..7952,7953..8021))
                     /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     7784..7803
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8022..8126)
                     /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 cgtcgaagtc gaagtcgacc
      901 tcccgtctca gtaaaggtcg tcgaagtcga agtcgaccaa tcggactgcc ttcgtacgtc
      961 gaagtcgaag tcgaccgtat cagtcgcctc ggaacgtcga agtcgaagtc gaccattcgt
     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 gcggggcggg cgcccgaagg tcctccggag gcccggcatt
     3121 ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct
     3181 ttcgatatct cgcacagtgc cgccaccatg accgagtaca agcccacggt gcgcctcgcc
     3241 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     3301 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     3361 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     3421 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     3481 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     3541 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     3601 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     3661 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     3721 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     3781 tgcatgaccc gcaagcccgg tgccggaagc ggagctacta acttcagcct gctgaagcag
     3841 gctggagacg tggaggagaa ccctggacct atgagcgagc tgattaagga gaacatgcac
     3901 atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc
     3961 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct
     4021 ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc
     4081 aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg
     4141 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc
     4201 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc
     4261 cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct
     4321 gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg
     4381 atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct
     4441 ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac
     4501 gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac
     4561 aagcttaatt aatctagata actgatcata atcagccata ccacatttgt agaggtttta
     4621 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     4681 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4741 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4801 aatgtatctt aggcgcgccc tgtccctagt ggccccactg tggggtggag gggacagata
     4861 aaagtaccca gaaccagagc cacattaacc ggccctggga atataaggtg gtcccagctc
     4921 ggggacacag gatccctgga ggcagcaaac atgctgtcct gaagtggaca taggggcccg
     4981 ggttggagga agaagactag ctgagctctc ggacccctgg aagatgccat gacagggggc
     5041 tggaagagct agcacagact agagaggtaa ggggggtagg ggagctgccc aaatgaaagg
     5101 agtgagaggt gacccgaatc cacaggagaa cggggtgtcc aggcaaagaa agcaagagga
     5161 tggagaggtg gctaaagcca gggagacggg gtactttggg gttgtccaga aaaacggtga
     5221 tgatgcaggc ctacaagaag gggaggcggg acgcaaggga gacatccgtc ggagaaggcc
     5281 atcctaagaa acgagagatg gcacaggccc cagaaggaga aggaaaaggg aacccagcga
     5341 gtgaagacgg catggggttg ggtgagggag gagagatgcc cggagaggac ccagacacgg
     5401 ggaggatccg ctcagaggac atcacgtggt gcagcgccga gaaggaagtg ctccggaaag
     5461 agcatccttg ggcagcaaca cagcagagag caaggggaag agggagtgga ggaagacgga
     5521 acctgaagga ggcggcaggg aaggatctgg gccagccgta gaggtgaccc aggccacaag
     5581 ctgcagacag aaagcggcac aggcccaggg gagagaatgc cctcgacctg cagcccaagc
     5641 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5701 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5761 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5821 cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     5881 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     5941 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     6001 ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata atggttacaa
     6061 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     6121 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca ttaatgaatc
     6181 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     6241 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6301 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6361 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6421 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6481 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6541 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6601 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6661 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6721 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6781 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6841 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6901 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6961 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7021 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7081 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7141 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7201 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7261 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7321 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7381 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7441 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7501 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7561 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7621 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7681 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7741 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7801 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7861 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7921 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7981 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8041 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8101 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tg
//
