LOCUS       sequence-304488-        5920 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 5920)
  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 5920)
  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..5920
                     /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"
     regulatory      1702..1711
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1708..1710,1711..1713,1714..2427)
                     /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(1753..1774)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2446..2462)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2446..2462)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2476..2493)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2482..2706
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2755..2772)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2755..2771)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2764..2786)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2912..3367)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3049..3070)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3261..3280
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3394..3498
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3499..3567,3568..4359)
                     /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(3717..3736)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4530..5118
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5019..5038
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5272..5289
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5406..5427
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5442..5447,5448..5465,5466..5472)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5480..5496
                     /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     5485..5507
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5504..5520
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5504..5520
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5545..5793
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5545..5565
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5716..5735
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5812..5887
                     /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 ccgggggggg gggggggggg sggggggsgg
      541 ggsggggcga 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 cttaattaag agtgcgatcg cgccaccatg gtgagcaagg gcgaggagct gttcaccggg
     1741 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgagg
     1801 ggcgagggcg agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc
     1861 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgagccacgg cgtgcagtgc
     1921 ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1981 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcacctacaa gacccgcgcc
     2041 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg cgtcgacttc
     2101 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaacatc
     2161 tatatcatgg ccgtcaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2221 gtggaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2281 ggccccgtgc tgctgcccga cagccactac ctgagcaccc agtccgtgct gagcaaagac
     2341 cccaacgaga agcgcgatca catggtcctg ctggagttcc gcaccgccgc cgggatcact
     2401 ctcggcatgg acgagctgta caagtgagac gcgtaaatga ttgcagatcc actagttcta
     2461 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2521 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2581 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2641 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggat gcggtgggct
     2701 ctatggcttc tgaggcggaa agaaccagct gggctcgagg ctagagcggc cgccactggc
     2761 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     2821 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     2881 ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa
     2941 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3001 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     3061 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3121 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3181 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3241 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3301 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3361 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3421 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3481 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3541 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     3601 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3661 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     3721 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     3781 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3841 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3901 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3961 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     4021 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     4081 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     4141 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     4201 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     4261 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4321 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4381 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4441 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4501 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4561 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4621 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4681 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4741 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4801 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4861 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4921 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4981 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5041 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5101 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5161 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5221 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     5281 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     5341 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     5401 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     5461 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     5521 catgaggcgc gccccccctt caccgagggc ctatttccca tgattccttc atatttgcat
     5581 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
     5641 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     5701 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     5761 gctttatata tcttgtggaa aggacgaaac accgggtctt cgagaagacc tgttttagag
     5821 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     5881 tcggtgcttt ttttgatcca agggtggggc gcgccggatt
//
