LOCUS       sequence-291977-        5916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5916)
  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 5916)
  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-BFP-pA_U6-BbsI (pE058)
FEATURES             Location/Qualifiers
     source          1..5916
                     /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"
     regulatory      1697..1706
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1703..1705,1706..1708,1709..2422)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1748..1769)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2441..2457)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2441..2457)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2471..2488)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2477..2701
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2750..2767)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2750..2766)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2759..2781)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2907..3362)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3044..3065)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3256..3275
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3389..3493
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3494..3562,3563..4354)
                     /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(3712..3731)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4525..5113
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5014..5033
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5267..5284
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5401..5422
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5437..5442,5443..5460,5461..5467)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5475..5491
                     /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     5480..5502
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5499..5515
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5499..5515
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5541..5789
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5541..5561
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5712..5731
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5808..5883
                     /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 ttaagagtgc gatcgcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     1741 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgaggggcga
     1801 gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1861 gctgcccgtg ccctggccca ccctcgtgac caccctgagc cacggcgtgc agtgcttcgc
     1921 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1981 cgtccaggag cgcaccatct tcttcaagga cgacggcacc tacaagaccc gcgccgaggt
     2041 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcgtcg acttcaagga
     2101 ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca acatctatat
     2161 catggccgtc aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacgtgga
     2221 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2281 cgtgctgctg cccgacagcc actacctgag cacccagtcc gtgctgagca aagaccccaa
     2341 cgagaagcgc gatcacatgg tcctgctgga gttccgcacc gccgccggga tcactctcgg
     2401 catggacgag ctgtacaagt gagacgcgta aatgattgca gatccactag ttctagagct
     2461 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2521 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2581 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2641 agcaaggggg aggattggga agagaatagc aggcatgctg gggatgcggt gggctctatg
     2701 gcttctgagg cggaaagaac cagctgggct cgaggctaga gcggccgcca ctggccgtcg
     2761 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2821 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2881 agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt taaaattcgc
     2941 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3001 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3061 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3121 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3181 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3241 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3301 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3361 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3421 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3481 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3541 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3601 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3661 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3721 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3781 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3841 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3901 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3961 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     4021 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     4081 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4141 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     4201 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4261 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     4321 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4381 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4441 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4501 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4561 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4621 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4681 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4741 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4801 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4861 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4921 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4981 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5041 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5101 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5161 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5221 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5281 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     5341 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     5401 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     5461 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     5521 ggcgcgcccc ccccttcacc gagggcctat ttcccatgat tccttcatat ttgcatatac
     5581 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     5641 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     5701 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     5761 tatatatctt gtggaaagga cgaaacaccg ggtcttcgag aagacctgtt ttagagctag
     5821 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     5881 tgcttttttt gatccaaggg tggggcgcgc cggatt
//
