LOCUS       sequence-246059-        5970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To integrate the CHYRON1 locus at HEK293site3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5970)
  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 5970)
  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: pTBL405 CHYRON1 integration construct
FEATURES             Location/Qualifiers
     source          1..5970
                     /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"
     promoter        1063..1311
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1063..1083
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1234..1253
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1447..1825
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1826..2029
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1980..2000
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2074..2093
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2074..2092
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2128..2727
                     /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(2128..2147)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2624..2644
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2887..3008
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2924..2943)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2978..2997
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3813..3829)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3813..3829)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3826..3848)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3837..3853
                     /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(3861..3867,3868..3885,3886..3891))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3906..3927
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4044..4061)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4215..4803)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4295..4314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4974..5765,5766..5834))
                     /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     5597..5616
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5835..5939)
                     /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 tgaattctct gtcacaggac tcagccaggc aagggggagg gagtgaaggg tgaggaggaa
      301 agagaggaca ttcagaacag gcgaactgca ggtgcaaagg cccggaatct agggaaagca
      361 gagtctgtgg gaggaacagc cagtgtttca ggatggctaa agcagaggtt gtgaagggca
      421 gggacgactt tagaccttag aggccccagg cactgttatt ttccaaggcc ccacccccac
      481 actataccat gcttattaaa attcacccac atcacacagg aaatatgagt tatatatggc
      541 tacctaaggt tgaggccagt tgcaaagaca attgacatgt tacattttgt agacatttaa
      601 ttacatattt atcaattaca acttgacggc tttcttgttt tatcagtttt ggaggatgta
      661 cataaatata tagaatgaga gtctcaaaac taatcttgta gctacgcctg tgatgggcta
      721 attgatgaat cagtgctgga gaatgggtca cagtggcaaa tgaggctgga gaggcccgtg
      781 agctgctgtc ccagagggcc tgggatgtcc tgcagggagc ttggcatgag aaaccttgga
      841 gagttttaag caagggctga tgtgggctgc ctagaaaggc atggatgaga gaagcctgga
      901 gacagggatc ccagggaaac gcccatgcaa ttagtctatt tctgctgcaa gtaagcatgc
      961 atttgtaggc ttgatgcttt tgacgtcgct agctgtacaa aaaagcaggc tttaaaggaa
     1021 ccaattcagt cgactggatc cggtaccaag gtcgggcagg aagagggcct atttcccatg
     1081 attccttcat atttgcatat acgatacaag gctgttagag agataattag aattaatttg
     1141 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     1201 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     1261 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cggcccagac
     1321 tgagcacgtg agggttagag ctagaaatag caagttaacc taaggctagt ccgttatcaa
     1381 cttgaaaaag tggcaccgag tcggtgcttt ttttaagctt gggccgctcg aggtacctct
     1441 ctacatacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     1501 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     1561 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     1621 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     1681 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     1741 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     1801 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     1861 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     1921 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     1981 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac
     2041 tagagaaccc actgcttact ggcttatcga aattaatacg actcactata gggagaccca
     2101 agctggctag cgctaccggt cgccaccatg accgagtaca agcccacggt gcgcctcgcc
     2161 acccgcgacg acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     2221 gccacgcgcc acaccgtcga cccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     2281 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     2341 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     2401 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     2461 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     2521 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     2581 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     2641 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     2701 tgcatgaccc gcaagcccgg tgcctgattc gaaatgaccg accaagcgac gcccaacctg
     2761 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     2821 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     2881 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2941 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3001 gtatcttatc atgtctgtat acagaggaaa ggaagccctg cttcctccag agggcgtcgc
     3061 aggacagctt ttcctagaca ggggctagta tgtgcagctc ctgcaccggg atactggttg
     3121 acaagtttgg ctgggctgga agccagcacc tagggaggtc cctggaaggg gccagcctca
     3181 ccaggagagg agggacctgg cccttcaggg tcgagctcaa cagaggaaaa gatctcaggg
     3241 cacccagagc ccagtggctt tcagcacctg catgaaaatc agagatcaac cagattaccc
     3301 catattgccc aagagaaaac tgaggccaga aagtgatgga gcttgcccag gaccccagag
     3361 ggtgttagag gcaggccaga accagaggac agctctgtcc cagcacttgc cctcctttaa
     3421 ttaggtgggt ggtcatatcg cagtggtcag agacattgca aacaataata ataaatattg
     3481 agcacttgtt acacgcaggg cactatgcta agcaccttgt atttgttatg tcctttcatc
     3541 ctagcaactt ctctggaagt ggagtaatat ctcccccatg tcctacataa gacaatggat
     3601 acactggagg gttaaagcca tgccttgcct aaagttacac agctgtgctt aacagctgga
     3661 aaaccccaaa gggtccctct gactcagccc ctgtgtgctc agttttaatt ttgggaggga
     3721 aataacaatg ccaccttata atactttcta agaatgagaa ttttgttcag tgaattcccg
     3781 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3841 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3901 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3961 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4021 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4081 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4141 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4201 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4261 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4321 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4381 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4441 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4501 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4561 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4621 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4681 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4741 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4801 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4861 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4921 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4981 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5041 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5101 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5161 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5221 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5281 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5341 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5401 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5461 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5521 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5581 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5641 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5701 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5761 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5821 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5881 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5941 acatttcccc gaaaagtgcc acctgacgtc
//
