LOCUS       sequence-291976-        5969 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 5969)
  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 5969)
  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..5969
                     /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..1672
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1596..1618
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1685..1704
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1685..1703
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1717..1737
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1738..1740,1741..1743,1744..2454)
                     /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(1783..1804)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2044..2063)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2391..2412
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2493..2509)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2493..2509)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2523..2540)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2529..2753
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2803..2820)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2803..2819)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2812..2834)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2960..3415)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3097..3118)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3309..3328
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3442..3546
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3547..3615,3616..4407)
                     /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(3765..3784)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4578..5166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5067..5086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5320..5337
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5454..5475
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5490..5495,5496..5513,5514..5520)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5528..5544
                     /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     5533..5555
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5552..5568
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5552..5568
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5594..5842
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5594..5614
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5765..5784
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5861..5936
                     /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 ccaggcgggg cggggcgggg cgaggggcgg
      541 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgtcgcgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      721 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      781 ccgggctgta attagcgctt ggtttaatga cggctcgttt cttttctgtg gctgcgtgaa
      841 agccttaaag ggctccggga gggccctttg tgcggggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggcccgcgc tgcccggcgg ctgtgagcgc
      961 tgcgggcgcg gcgcggggct ttgtgcgctc cgcgtgtgcg cgaggggagc gcggccgggg
     1021 gcggtgcccc gcggtgcggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg
     1081 cgtggggggg tgagcagggg gtgtgggcgc ggcggtcggg ctgtaacccc cccctgcacc
     1141 cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt gcggggcgtg
     1201 gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg
     1261 ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggccccgga gcgccggcgg
     1321 ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca
     1381 gggacttcct ttgtcccaaa tctggcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcgggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc
     1501 ttcgtgcgtc gccgcgccgc cgtccccttc tccatctcca gcctcggggc tgccgcaggg
     1561 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     1621 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agatccttaa
     1681 ttaataatac gactcactat aggggccgcc accatgccca agaagaagag gaaggtgatg
     1741 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1801 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1861 aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1921 gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag
     1981 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2041 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2101 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2161 ctggagtaca actacaacag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc
     2221 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac
     2281 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2341 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2401 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggcgga
     2461 ggcggaggcg gaggcacgcg taaatgattg cagatccact agttctagag ctcgctgatc
     2521 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2581 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2641 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2701 ggaggattgg gaagagaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2761 ggcggaaaga accagctggg gctcgaggct agagcggccg ccactggccg tcgttttaca
     2821 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2881 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2941 cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3001 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3061 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3121 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3181 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3241 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3301 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3361 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     3421 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3481 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3541 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3601 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3661 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3721 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3781 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3841 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3901 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3961 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4021 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4081 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4141 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4201 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4261 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4321 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4381 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4441 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4501 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4561 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4621 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4681 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4741 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4801 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4861 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4921 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4981 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5041 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5101 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5161 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5221 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5281 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5341 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5401 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     5461 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5521 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgaggcgcgc
     5581 cccccccttc accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     5641 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     5701 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     5761 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     5821 cttgtggaaa ggacgaaaca ccgggtcttc gagaagacct gttttagagc tagaaatagc
     5881 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     5941 tttgatccaa gggtggggcg cgccggatt
//
