LOCUS       sequence-490973-        5244 bp    DNA     circular SYN 29-OCT-2025
DEFINITION  for HEK293T human cell genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5244)
  AUTHORS   Jin S, Zhu Z, Li Y, Zhang S, Liu Y, Li D, Li Y, Luo Y, Cheng Z, Zhao
            KT, Gao Q, Yang G, Li H, Liang R, Zhang R, Qiu JL, Zhang YE, Liu JG,
            Gao C
  TITLE     Functional RNA splitting drove the evolutionary emergence of type V 
            CRISPR-Cas systems from transposons.
  JOURNAL   Cell. 2025 Sep 29:S0092-8674(25)01035-9. doi: 
            10.1016/j.cell.2025.09.004.
  PUBMED    41027434
REFERENCE   2  (bases 1 to 5244)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 29, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2025 
            Sep 29:S0092-8674(25)01035-9. doi: 10.1016/j.cell.2025.09.004."
COMMENT     Sequence Label: pCMV-eLaTranC
FEATURES             Location/Qualifiers
     source          1..5244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2230..2250
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2259..2276
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2299..2316)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2305..2529
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2600..2616)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2600..2616)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2613..2635)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2624..2640
                     /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(2648..2654,2655..2672,2673..2678))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2693..2714
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2831..2848)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3002..3590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3082..3101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3761..4552,4553..4621))
                     /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     4384..4403
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4622..4726)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4805..4824)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(4996..5244,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcagcgt gtcttttagc
      481 ctgaatgcca aaaagattcg gctggaaaac tacgccatga aaatgagact ctacccttct
      541 cctacacagg ccgaacagat ggacaagatg ttcctggccc tgagactggc ctacaacatg
      601 accttccacg aagtgtttca gcagaaccct gccgtgtgcg gcgaccccga cgaggacggc
      661 aacgtgtggc ctagctacaa gaagatggcc aataagacct ggcggaaggc tctgatcgac
      721 cagaatccag ccatcgccga ggctcccgca gccgccatca ccaccaacaa cggactgttc
      781 ctgtccaacg gccagaaggc ctggaagacc ggcatgcaca acctgccagc caacaaggct
      841 gatagaaagg actttaggtt ttatagcctg cgtaagccca gaagaagctt cgccgtgcag
      901 atccgtccac gctgcatcat ccccagcgat accaaccaga aggtggccag aatcaagctg
      961 cctaagatcg acggcgccat caaggccaga ggcttcaacc ggaagatctg gttcggcccc
     1021 gacggaaaac atacatacga ggaagctctg gccgcccacg agctgtccaa caacctgacc
     1081 gtgcgggtgt ccaaagatac atgcggcgat tacttcatct gcatcacctt ctcccagggc
     1141 aaggtgaagg gcgacaagcc tacctgggag ttctaccagg aggtgagagt cagccctatc
     1201 cccgagccca tcggcctgga cgtgggcatc aaggacatcg ccatcctgaa tacaggcaca
     1261 aagtacgaga acaagcagtt caagcgggac agagccgcaa ccctgaaaaa gatgagccgg
     1321 cagctgagca gaagatgggg ccctgccaac agcgctttcc gggactacaa caaaaacatc
     1381 cgggccgaga acagagccct ggaaagagct caacaggacc ctggctcttc cggcgtggga
     1441 cctgaggccc ctgtgctcaa gtctgtggcc caacctagca gaagatacct gaccatccag
     1501 aaaaacagag ccaagctgga acgcaagatc gccagaagac gcgataccta ctaccaccag
     1561 gtgacccgcg aggtggccgg aaagagcagc ctgctggccg tggaaaccct gcgggtcaag
     1621 aacatgctgc agaaccacag actggccttc gcactgtctg atgctgctat gagcgacttc
     1681 atcagcaagc tgaagtataa ggctcgaaga atccaggttc ctctggtggc catcggcacc
     1741 tttcagccca gcagccagac atgtagcgtg tgcggctcca tcaaccccgc tgttaagaac
     1801 ctgtctatca gagtgtggac ctgtcctaac tgtggaacaa gacacaatcg ggatatcaat
     1861 gccgctaaga acatcctggc tatcgcacaa aacatgctgg aaaagaaggt gcccttcgcc
     1921 gacgaggccc tgccagatga gaagcctcct gccgctccag tgaagaaggc cgcccggaaa
     1981 cctagggacg ccgtgttccc tgatcaccct gacctggtca tcagattcag caaagagctg
     2041 acacagctca acgatcctag atacgtgatc gtgaacaagg ccaccaatca gattgtggac
     2101 aacgcccaag gcgccggcta ccggagcgcc gccaaagcca agaactgcta caaagccaag
     2161 ctggcctgga gcagcaagac aaacaagtct ggcggctcaa aaagaaccgc cgacggcagc
     2221 gaattcgagc ccaagaagaa gaggaaagtc taaccggtca tcatcaccat caccattgag
     2281 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2341 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2401 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2461 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     2521 gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg acctctagct
     2581 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2641 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga
     2701 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2761 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2821 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2881 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2941 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3001 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3061 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3121 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3181 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3241 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3301 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3361 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3421 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3481 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3541 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3601 tgatcttttc tacggggtct gacactcagt ggaacgaaaa ctcacgttaa gggattttgg
     3661 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3721 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3781 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3841 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3901 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3961 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4021 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4081 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4141 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4201 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4261 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4321 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4381 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4441 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4501 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4561 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4621 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4681 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4741 aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc
     4801 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     4861 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     4921 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     4981 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     5041 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     5101 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     5161 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     5221 acttggcagt acatcaagtg tatc
//
