LOCUS       sequence-305313-        5972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for Venus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5972)
  AUTHORS   Danner E, Lebedin M, de la Rosa K, Kuhn R
  TITLE     A homology independent sequence replacement strategy in human cells 
            using a CRISPR nuclease.
  JOURNAL   Open Biol. 2021 Jan;11(1):200283. doi: 10.1098/rsob.200283. Epub 
            2021 Jan 27.
  PUBMED    33499763
REFERENCE   2  (bases 1 to 5972)
  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"; journalName: "Open 
            Biol."; date: "2021-01"; volume: "11(1):200283. doi"; pages: " 
            10.1098/rsob.200283. Epub 2021 Jan 27"
COMMENT     Sequence Label: pCAG-Venus-pA_U6-BbsI (pE057)
FEATURES             Location/Qualifiers
     source          1..5972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        2..381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        383..660
                     /label=chicken beta-actin promoter
     intron          661..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1601..1623
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1690..1709
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1690..1708
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1722..1742
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1743..1745,1746..1748,1749..2459)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1788..1809)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2049..2068)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2396..2417
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2498..2514)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2498..2514)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2528..2545)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2534..2758
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2807..2824)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2807..2823)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2816..2838)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2964..3419)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3101..3122)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3313..3332
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3446..3550
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3551..3619,3620..4411)
                     /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     complement(3769..3788)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4582..5170
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5071..5090
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5324..5341
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5458..5479
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5494..5499,5500..5517,5518..5524)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5532..5548
                     /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     5537..5559
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5556..5572
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5556..5572
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5597..5845
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5597..5617
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5768..5787
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5864..5939
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
       61 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      121 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      181 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      241 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      301 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      361 ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc
      421 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
      481 atgggggcgg gggggggggg ggggcgcgcg ccrggggggg gggggggggg gggggggsgg
      541 ggsggggsgr ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      721 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      781 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      841 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      901 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      961 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1261 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1561 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagatc
     1681 cttaattaat aatacgactc actatagggg ccgccaccat gcccaagaag aagaggaagg
     1741 tgatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1801 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1861 acggcaagct gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg ccctggccca
     1921 ccctcgtgac caccctgggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga
     1981 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2041 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2101 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2161 acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga
     2221 acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg
     2281 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2341 actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     2401 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     2461 gcggaggcgg aggcggaggc acgcgtaaat gattgcagat ccactagttc tagagctcgc
     2521 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2581 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2641 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2701 aagggggagg attgggaaga gaatagcagg catgctgggg atgcggtggg ctctatggct
     2761 tctgaggcgg aaagaaccag ctgggctcga ggctagagcg gccgccactg gccgtcgttt
     2821 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2881 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2941 tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat attttgttaa aattcgcgtt
     3001 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     3061 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     3121 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     3181 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     3241 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     3301 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     3361 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     3421 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3481 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3541 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3601 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3661 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3721 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3781 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3841 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3901 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3961 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4021 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4081 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4141 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4201 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4261 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4321 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4381 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4441 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4501 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4561 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4621 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4681 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4741 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4801 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4861 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4921 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4981 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5041 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5101 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5161 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5221 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5281 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     5341 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5401 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5461 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5521 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgaggc
     5581 gcgccccccc ttcaccgagg gcctatttcc catgattcct tcatatttgc atatacgata
     5641 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     5701 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     5761 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     5821 tatcttgtgg aaaggacgaa acaccgggtc ttcgagaaga cctgttttag agctagaaat
     5881 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     5941 ttttttgatc caagggtggg gcgcgccgga tt
//
