LOCUS       sequence-225521-        8805 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mTagBFP in human cells, contained in a lentiviral vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8805)
  AUTHORS   Watters KE, Fellmann C, Bai HB, Ren SM, Doudna JA
  TITLE     Systematic discovery of natural CRISPR-Cas12a inhibitors.
  JOURNAL   Science. 2018 Oct 12;362(6411):236-239. doi: 
            10.1126/science.aau5138. Epub 2018 Sep 6.
  PUBMED    30190307
REFERENCE   2  (bases 1 to 8805)
  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"; doi: 
            "10.1126/science.aau5138"; journalName: "Science"; date: 
            "2018-10-12- 12"; volume: "362"; issue: "6411"; pages: "236-239"
COMMENT     Sequence Label: pCF525-EF1a-Hygro-P2A-mTagBFP2-lenti
FEATURES             Location/Qualifiers
     source          1..8805
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        117..496
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        498..696
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     651..671
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             714..894
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    941..1066
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1559..1792
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1977..2021
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2319..2436
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2511..3688
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2742..3679
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3636..3656
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3706..3715
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3712..4737
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MAKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4747..4803
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     misc_feature    5512..6100
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5565..5585)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5983..5994)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6172..6405
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6431..6448)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6437..6661
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6767..6784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6938..7526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7018..7037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7697..8488,8489..8557))
                     /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     8320..8339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8558..8662)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8737..8756)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
       61 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatattgaca
      121 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      181 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      241 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      301 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      361 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      421 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      481 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      541 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      601 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      661 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      721 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      781 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      841 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
      901 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
      961 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1021 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     1081 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     1141 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     1201 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     1261 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     1321 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     1381 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
     1441 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     1501 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     1561 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     1621 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     1681 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     1741 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     1801 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     1861 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     1921 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     1981 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     2041 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     2101 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     2161 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag
     2221 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg
     2281 cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     2341 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     2401 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     2461 gatccagttt ggttaattaa gctagctagg tcttgaaagg agtgggaatt ggctccggtg
     2521 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     2581 gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     2641 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     2701 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
     2761 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccactg
     2821 gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag
     2881 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
     2941 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc
     3001 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct
     3061 tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga
     3121 cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc
     3181 gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc
     3241 gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
     3301 ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc
     3361 gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt
     3421 cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag
     3481 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca
     3541 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga
     3601 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag
     3661 tttttttctt ccatttcagg tgtcgtgaac cggttctaga gcgctgccac catggccaaa
     3721 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtg
     3781 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     3841 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     3901 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggag
     3961 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     4021 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggcaat ggatgcgatc
     4081 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     4141 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     4201 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     4261 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     4321 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     4381 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     4441 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     4501 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     4561 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     4621 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     4681 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaagga
     4741 tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga gaatcctgga
     4801 ccgtctgaac tcatcaagga gaacatgcac atgaaactct acatggaggg gaccgtagac
     4861 aatcatcact tcaagtgtac aagtgaaggg gaggggaagc cttacgaagg gactcaaact
     4921 atgagaatta aggttgtcga gggcggcccc cttccctttg cgtttgacat cctcgccacc
     4981 agtttcttgt atggttccaa gacgtttata aatcatacac agggaatacc tgacttcttt
     5041 aaacaaagtt ttccagaggg tttcacatgg gaaagggtta ccacttatga ggatggcgga
     5101 gtactcaccg ctacccaaga tacatctctg caagatggtt gcctgattta caatgtaaaa
     5161 atccgaggag ttaactttac atccaatggt cctgttatgc agaaaaagac attggggtgg
     5221 gaagcattta cagaaacgct ctaccccgcc gacgggggct tggagggccg caatgacatg
     5281 gctctcaaat tggtaggtgg tagtcatctt atcgcgaacg cgaaaactac ctaccgatcc
     5341 aagaagcccg ctaaaaatct caaaatgccg gtctattacg tggactacag actggaacgc
     5401 aaggaagcta acaacgagac atacgttgag caacatgaag tggccgtggc cagatattgc
     5461 gatctgccga gtaagctcgg gcacaaattg aactaaacgc gttaagtcga caatcaacct
     5521 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     5581 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     5641 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     5701 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     5761 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     5821 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     5881 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     5941 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     6001 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     6061 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
     6121 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     6181 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     6241 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     6301 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     6361 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc
     6421 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     6481 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     6541 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     6601 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     6661 gcttctgagg cggaaagaac cagccattaa tgaaaagaac cagccattaa tgaatcggcc
     6721 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     6781 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6841 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6901 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6961 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     7021 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     7081 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     7141 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     7201 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     7261 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     7321 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     7381 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     7441 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     7501 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     7561 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7621 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7681 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     7741 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7801 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7861 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7921 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7981 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     8041 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     8101 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     8161 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     8221 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     8281 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     8341 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     8401 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     8461 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     8521 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     8581 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     8641 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     8701 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     8761 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctga
//
