LOCUS       sequence-246061-        6642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To integrate the CHYRON3 locus at AAVS1 in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6642)
  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 6642)
  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: pTBL680 CHYRON3 integration construct
FEATURES             Location/Qualifiers
     source          1..6642
                     /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        1732..2110
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2111..2314
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2265..2285
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2359..2378
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2359..2377
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2413..3012
                     /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(2413..2432)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2909..2929
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3172..3293
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3209..3228)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3263..3282
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    3606..4135
                     /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(4485..4501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4485..4501)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4498..4520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4509..4525
                     /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(4533..4539,4540..4557,4558..4563))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4578..4599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4716..4733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4887..5475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4967..4986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5646..6437,6438..6506))
                     /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     6269..6288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6507..6611)
                     /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 acaccggccc agactgagca cgtgagggtt agagctagaa atagcaagtt
     1621 aacctaaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttcta
     1681 gacccagcaa ttgtgagcgc tcacaatgcc gctcgaggta cctctctaca tacattgatt
     1741 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1801 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1861 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1921 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1981 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     2041 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     2101 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     2161 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     2221 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     2281 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
     2341 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg gctagcgcta
     2401 ccggtcgcca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     2461 ccccgggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     2521 gtcgacccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     2581 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     2641 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     2701 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     2761 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     2821 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     2881 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     2941 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     3001 cccggtgcct gattcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     3061 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     3121 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     3181 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3241 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     3301 tgtataagct agtcaggtag taaaaggtct caactaggca gtggcagagc aggattcaaa
     3361 ttcagggctg ttgtgatgcc tccgcagact ctgagcgcca cctggtggta atttgtctgt
     3421 gcctcttctg acgtggaaga acagcaacta acacactaac acggcattta ctatgggcca
     3481 gccattgttc taattgggtc taacccccac ctcctgttag gcagattcct tatctggtga
     3541 cacaccccca tttcctggag ccatctctct ccttgccaga acctctaagg tttgcttacg
     3601 atggagccag agaggatcct gggagggaga gcttggcagg gggtgggagg gaaggggggg
     3661 atgcgtgacc tgcccggttc tcagtggcca ccctgcgcta ccctctccca gaacctgagc
     3721 tgctctgacg cggctgtctg gtgcgtttca ctgatcctgg tgctgcagct tccttacact
     3781 tcccaagagg agaagcagtt tggaaaaaca aaatcagaat aagttggtcc tgagttctaa
     3841 ctttggctct tcacctttct agtccccaat ttatattgtt cctccgtgcg tcagttttac
     3901 ctgtgagata aggccagtag ccagccccgt cctggcaggg ctgtggtgag gaggggggtg
     3961 tccgtgtgga aaactccctt tgtgagaatg gtgcgtccta ggtgttcacc aggtcgtggc
     4021 cgcctctact ccctttctct ttctccatcc ttctttcctt aaagagtccc cagtgctatc
     4081 tgggacatat tcctccgccc agagcagggt cccgcttccc taaggccctg ctctgggctt
     4141 ctgggtttga gtccttggca agcccaggag aggcgctcag gcttccctgt cccccttcct
     4201 cgtccaccat ctcatgcccc tggctctcct gccccttccc tacaggggtt cctggctctg
     4261 ctcttcagac tgagccccgt tcccctgcat ccccgttccc ctgcatcccc cttcccctgc
     4321 atcccccaga ggccccaggc cacctacttg gcctggaccc cacgagaggc caccccagcc
     4381 ctgtctacca ggctgccttt tgggtggatt ctcctccaac tgtggggtga ctgcttggca
     4441 aacgaattcc cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4501 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4561 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4621 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4681 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4741 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4801 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4861 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4921 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4981 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5041 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5101 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5161 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5221 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5281 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5341 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5401 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5461 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5521 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5581 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5641 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5701 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5761 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5821 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5881 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5941 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6001 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6061 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6121 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6181 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6241 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6301 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6361 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6421 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6481 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6541 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6601 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
