LOCUS       sequence-246016-        6638 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To integrate the CHYRON4 locus at AAVS1 in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6638)
  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 6638)
  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: pTBL681 CHYRON4 integration construct
FEATURES             Location/Qualifiers
     source          1..6638
                     /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"
     primer_bind     1326..1346
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     protein_bind    1435..1451
                     /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)."
     primer_bind     1497..1516
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    1528..1547
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     enhancer        1728..2106
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2107..2310
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2261..2281
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2355..2374
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2355..2373
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2409..3008
                     /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(2409..2428)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2905..2925
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3168..3289
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3205..3224)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3259..3278
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    3602..4131
                     /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(4481..4497)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4481..4497)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4494..4516)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4505..4521
                     /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(4529..4535,4536..4553,4554..4559))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4574..4595
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4712..4729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4883..5471)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4963..4982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5642..6433,6434..6502))
                     /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     6265..6284
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6503..6607)
                     /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 cttaggcagc tagtcaggta gtaaaaggtc
     1021 tcaactaggc agtggcagag caggattcaa attcagggct gttgtgatgc ctccgcagac
     1081 tctgagcgcc acctggtggt aatttgtctg tgcctcttct gacgtggaag aacagcaact
     1141 aacacactaa cacggcattt actatgggcc agccattgtt ctaaagccct cgttcacaga
     1201 gaaagttcgg ttacactgcg gattgaggcg gacactgatt gacacggttt gctagctgta
     1261 caaaaaagca ggctttaaag gaaccaattc agtcgactgg atccggtacc aaggtcgggc
     1321 aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     1381 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aaaattgtga
     1441 gcggataaca attatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     1501 atcatatgct taccgtaact tgaaagtaat tgtgagcgct cacaattata tatcttgtgg
     1561 aaaggacgaa acaccgagac tgagcacgtg agggttagag ctagaaatag caagttaacc
     1621 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttctagacc
     1681 cagcaattgt gagcgctcac aatgccgctc gaggtacctc tctacataca ttgattattg
     1741 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     1801 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     1861 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     1921 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     1981 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     2041 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     2101 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     2161 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     2221 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     2281 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac
     2341 tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta gcgctaccgg
     2401 tcgccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     2461 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     2521 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     2581 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     2641 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     2701 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     2761 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     2821 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     2881 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     2941 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     3001 gtgcctgatt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3061 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3121 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     3181 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3241 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3301 taagctagtc aggtagtaaa aggtctcaac taggcagtgg cagagcagga ttcaaattca
     3361 gggctgttgt gatgcctccg cagactctga gcgccacctg gtggtaattt gtctgtgcct
     3421 cttctgacgt ggaagaacag caactaacac actaacacgg catttactat gggccagcca
     3481 ttgttctaat tgggtctaac ccccacctcc tgttaggcag attccttatc tggtgacaca
     3541 cccccatttc ctggagccat ctctctcctt gccagaacct ctaaggtttg cttacgatgg
     3601 agccagagag gatcctggga gggagagctt ggcagggggt gggagggaag ggggggatgc
     3661 gtgacctgcc cggttctcag tggccaccct gcgctaccct ctcccagaac ctgagctgct
     3721 ctgacgcggc tgtctggtgc gtttcactga tcctggtgct gcagcttcct tacacttccc
     3781 aagaggagaa gcagtttgga aaaacaaaat cagaataagt tggtcctgag ttctaacttt
     3841 ggctcttcac ctttctagtc cccaatttat attgttcctc cgtgcgtcag ttttacctgt
     3901 gagataaggc cagtagccag ccccgtcctg gcagggctgt ggtgaggagg ggggtgtccg
     3961 tgtggaaaac tccctttgtg agaatggtgc gtcctaggtg ttcaccaggt cgtggccgcc
     4021 tctactccct ttctctttct ccatccttct ttccttaaag agtccccagt gctatctggg
     4081 acatattcct ccgcccagag cagggtcccg cttccctaag gccctgctct gggcttctgg
     4141 gtttgagtcc ttggcaagcc caggagaggc gctcaggctt ccctgtcccc cttcctcgtc
     4201 caccatctca tgcccctggc tctcctgccc cttccctaca ggggttcctg gctctgctct
     4261 tcagactgag ccccgttccc ctgcatcccc gttcccctgc atcccccttc ccctgcatcc
     4321 cccagaggcc ccaggccacc tacttggcct ggaccccacg agaggccacc ccagccctgt
     4381 ctaccaggct gccttttggg tggattctcc tccaactgtg gggtgactgc ttggcaaacg
     4441 aattcccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4501 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4561 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4621 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4681 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4741 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4801 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4861 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4921 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4981 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5041 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5101 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5161 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5221 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5281 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5341 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5401 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5461 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5521 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5581 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5641 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5701 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5761 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5821 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5881 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5941 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6001 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6061 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6121 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6181 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6241 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6301 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6361 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6421 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6481 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6541 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6601 ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
