LOCUS       sequence-384883-        8153 bp    DNA     circular SYN 15-APR-2025
DEFINITION  AAVS1 Integratable mCherry Reporter for synZiFTR ZF6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8153)
  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 8153)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-014
FEATURES             Location/Qualifiers
     source          1..8153
                     /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     1094..1118
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1141..1150
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1147..1857
                     /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(1292..1310)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1560..1579
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1943..1962)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2008..2063
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2062..2081)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    2409..2633
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2698..3185
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2733..2756)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3118..3137
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3209..3805
                     /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(3209..3228)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3705..3725
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3815..3871
                     /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             3872..4573
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4691..4812
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    4953..5454
                     /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(5656..5672)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5656..5672)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5669..5691)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5680..5696
                     /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(5704..5710,5711..5728,5729..5734))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5749..5770
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5829..6024
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5875..6010
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6030..6164
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6232..6249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6403..6991)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6483..6502)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7162..7953,7954..8022))
                     /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     7785..7804
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8023..8127)
                     /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 atccgccggc gaattcgtcg
      841 accgggtttc gtaacaatcg catgaggatt cgcaacgcct tggacgacgc ggtctaagaa
      901 gtcccgtctc agtaaaggtg gacgacgcgg tctaagaaga atcggactgc cttcgtagga
      961 cgacgcggtc taagaaggta tcagtcgcct cggaaggacg acgcggtcta agaagattcg
     1021 taagaggctc actctccctt acacggagtg gataactagt taggcgtgta cggtgggagg
     1081 cctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggaacgcg taccggtgtc
     1141 gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     1201 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1261 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     1321 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     1381 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     1441 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     1501 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1561 cccgtaatgc agaagaagac catgggctgg caggcctcct ccgagcggat gtaccccgag
     1621 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     1681 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     1741 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     1801 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaagcg
     1861 gccgcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     1921 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     1981 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     2041 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     2101 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     2161 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     2221 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     2281 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     2341 tctcctgact actcccagtc atagctgtcc ctcttctctt atgaagatcc ctcgacctgc
     2401 agggtaccct gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2461 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2521 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2581 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2641 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcgtaccggg
     2701 taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca
     2761 cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc
     2821 aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga
     2881 agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc
     2941 tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg
     3001 gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg
     3061 ggtccggggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     3121 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     3181 tttcgatatc tcgcacagtg ccgccaccat gaccgagtac aagcccacgg tgcgcctcgc
     3241 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     3301 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     3361 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     3421 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     3481 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     3541 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     3601 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     3661 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     3721 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     3781 gtgcatgacc cgcaagcccg gtgccggaag cggagctact aacttcagcc tgctgaagca
     3841 ggctggagac gtggaggaga accctggacc tatgagcgag ctgattaagg agaacatgca
     3901 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
     3961 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     4021 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
     4081 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
     4141 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     4201 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     4261 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc
     4321 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     4381 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     4441 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgagaccta
     4501 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     4561 caagcttaat taatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     4621 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     4681 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4741 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4801 caatgtatct taggcgcgcc ctgtccctag tggccccact gtggggtgga ggggacagat
     4861 aaaagtaccc agaaccagag ccacattaac cggccctggg aatataaggt ggtcccagct
     4921 cggggacaca ggatccctgg aggcagcaaa catgctgtcc tgaagtggac ataggggccc
     4981 gggttggagg aagaagacta gctgagctct cggacccctg gaagatgcca tgacaggggg
     5041 ctggaagagc tagcacagac tagagaggta aggggggtag gggagctgcc caaatgaaag
     5101 gagtgagagg tgacccgaat ccacaggaga acggggtgtc caggcaaaga aagcaagagg
     5161 atggagaggt ggctaaagcc agggagacgg ggtactttgg ggttgtccag aaaaacggtg
     5221 atgatgcagg cctacaagaa ggggaggcgg gacgcaaggg agacatccgt cggagaaggc
     5281 catcctaaga aacgagagat ggcacaggcc ccagaaggag aaggaaaagg gaacccagcg
     5341 agtgaagacg gcatggggtt gggtgaggga ggagagatgc ccggagagga cccagacacg
     5401 gggaggatcc gctcagagga catcacgtgg tgcagcgccg agaaggaagt gctccggaaa
     5461 gagcatcctt gggcagcaac acagcagaga gcaaggggaa gagggagtgg aggaagacgg
     5521 aacctgaagg aggcggcagg gaaggatctg ggccagccgt agaggtgacc caggccacaa
     5581 gctgcagaca gaaagcggca caggcccagg ggagagaatg ccctcgacct gcagcccaag
     5641 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5701 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5761 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5821 gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     5881 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     5941 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     6001 tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca
     6061 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     6121 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat
     6181 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     6241 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6301 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6361 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6421 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6481 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6541 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6601 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6661 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6721 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6781 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6841 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6901 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6961 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     7021 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     7081 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     7141 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     7201 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     7261 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     7321 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7381 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7441 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7501 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7561 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7621 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7681 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7741 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7801 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7861 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7921 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7981 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     8041 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     8101 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
