LOCUS       sequence-246053-        6182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To integrate the CHYRON2 locus at AAVS1 in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6182)
  AUTHORS   Loveless TB, Grotts JH, Schechter MW, Forouzmand E, Carlson CK, 
            Agahi BS, Liang G, Ficht M, Liu B, Xie X, Liu CC
  TITLE     Lineage tracing and analog recording in mammalian cells by 
            single-site DNA writing.
  JOURNAL   Nat Chem Biol. 2021 Jun;17(6):739-747. doi: 
            10.1038/s41589-021-00769-8. Epub 2021 Mar 22.
  PUBMED    33753928
REFERENCE   2  (bases 1 to 6182)
  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: 
            "10.1038/s41589-021-00769-8"; journalName: "Nat Chem Biol"; date: 
            "2021-06"; volume: "17"; issue: "6"; pages: "739-747"
COMMENT     Sequence Label: pTBL649 CHYRON2 integration construct
FEATURES             Location/Qualifiers
     source          1..6182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    333..834
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        1079..1327
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1079..1099
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1250..1269
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1459..1837
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1838..2041
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1992..2012
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2086..2105
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2086..2104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2140..2739
                     /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(2140..2159)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2636..2656
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2899..3020
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2936..2955)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2990..3009
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    3146..3675
                     /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     complement(4025..4041)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4025..4041)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4038..4060)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4049..4065
                     /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(4073..4079,4080..4097,4098..4103))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4118..4139
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4256..4273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4427..5015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4507..4526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5186..5977,5978..6046))
                     /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     5809..5828
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6047..6151)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttggtgag
      241 tgaattcctg ccgcctcctt caggttccgt cttcctccac tccctcttcc ccttgctctc
      301 tgctgtgttg ctgcccaagg atgctctttc cggagcactt ccttctcggc gctgcaccac
      361 gtgatgtcct ctgagcggat cctccccgtg tctgggtcct ctccgggcat ctctcctccc
      421 tcacccaacc ccatgccgtc ttcactcgct gggttccctt ttccttctcc ttctggggcc
      481 tgtgccatct ctcgtttctt aggatggcct tctccgacgg atgtctccct tgcgtcccgc
      541 ctccccttct tgtaggcctg catcatcacc gtttttctgg acaaccccaa agtaccccgt
      601 ctccctggct ttagccacct ctccatcctc ttgctttctt tgcctggaca ccccgttctc
      661 ctgtggattc gggtcacctc tcactccttt catttgggca gctcccctac cccccttacc
      721 tctctagtct gtgctagctc ttccagcccc ctgtcatggc atcttccagg ggtccgagag
      781 ctcagctagt cttcttcctc caacccgggc ccctatgtcc acttcaggac agcatgtttg
      841 ctgcctccag ggatcctgtg tccccgagct gggaccacct tatattccca gggccggtta
      901 atgtggctct ggttctgggt acttttatct gtcccctcca ccccacagtg gggccactag
      961 ggacaggatt ggtgacagaa aagccccatc cttaggcgac gtcgctagct gtacaaaaaa
     1021 gcaggcttta aaggaaccaa ttcagtcgac tggatccggt accaaggtcg ggcaggaaga
     1081 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     1141 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     1201 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     1261 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     1321 aaacaccgag actgagcacg tgagggttag agctagaaat agcaagttaa cctaaggcta
     1381 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttaagc ttgggccgct
     1441 cgaggtacct ctctacatac attgattatt gactagttat taatagtaat caattacggg
     1501 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     1561 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     1621 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1681 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     1741 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     1801 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     1861 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     1921 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     1981 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     2041 tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta
     2101 tagggagacc caagctggct agcgctaccg gtcgccacca tgaccgagta caagcccacg
     2161 gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc
     2221 gccgactacc ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc
     2281 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     2341 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtagaagc gggggcggtg
     2401 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     2461 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     2521 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     2581 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     2641 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     2701 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgat tcgaaatgac cgaccaagcg
     2761 acgcacaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     2821 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     2881 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     2941 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3001 aaactcatca atgtatctta tcatgtctgt atattgggtc taacccccac ctcctgttag
     3061 gcagattcct tatctggtga cacaccccca tttcctggag ccatctctct ccttgccaga
     3121 acctctaagg tttgcttacg atggagccag agaggatcct gggagggaga gcttggcagg
     3181 gggtgggagg gaaggggggg atgcgtgacc tgcccggttc tcagtggcca ccctgcgcta
     3241 ccctctccca gaacctgagc tgctctgacg cggctgtctg gtgcgtttca ctgatcctgg
     3301 tgctgcagct tccttacact tcccaagagg agaagcagtt tggaaaaaca aaatcagaat
     3361 aagttggtcc tgagttctaa ctttggctct tcacctttct agtccccaat ttatattgtt
     3421 cctccgtgcg tcagttttac ctgtgagata aggccagtag ccagccccgt cctggcaggg
     3481 ctgtggtgag gaggggggtg tccgtgtgga aaactccctt tgtgagaatg gtgcgtccta
     3541 ggtgttcacc aggtcgtggc cgcctctact ccctttctct ttctccatcc ttctttcctt
     3601 aaagagtccc cagtgctatc tgggacatat tcctccgccc agagcagggt cccgcttccc
     3661 taaggccctg ctctgggctt ctgggtttga gtccttggca agcccaggag aggcgctcag
     3721 gcttccctgt cccccttcct cgtccaccat ctcatgcccc tggctctcct gccccttccc
     3781 tacaggggtt cctggctctg ctcttcagac tgagccccgt tcccctgcat ccccgttccc
     3841 ctgcatcccc cttcccctgc atcccccaga ggccccaggc cacctacttg gcctggaccc
     3901 cacgagaggc caccccagcc ctgtctacca ggctgccttt tgggtggatt ctcctccaac
     3961 tgtggggtga ctgcttggca aacgaattcc cgtcgacctc tagctagagc ttggcgtaat
     4021 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4081 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4141 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4201 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4261 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4321 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4381 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4441 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4501 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4561 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4621 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4681 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4741 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4801 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4861 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4921 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4981 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5041 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5101 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5161 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5221 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5281 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5341 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5401 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5461 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5521 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5581 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5641 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5701 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5761 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5821 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5881 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5941 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6001 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6061 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6121 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6181 tc
//
