LOCUS       sequence-299807-        6141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for miRFP670.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6141)
  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 6141)
  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-miRFP670-pA_U6-BbsI (pE059)
FEATURES             Location/Qualifiers
     source          1..6141
                     /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      1693..1702
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1699..2646
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     primer_bind     complement(2667..2683)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2667..2683)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2697..2714)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2703..2927
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2976..2993)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2976..2992)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2985..3007)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3133..3588)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3270..3291)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3482..3501
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3615..3719
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3720..3788,3789..4580)
                     /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(3938..3957)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4751..5339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5240..5259
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5493..5510
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5627..5648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5663..5668,5669..5686,5687..5693)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5701..5717
                     /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     5706..5728
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5725..5741
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5725..5741
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5766..6014
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5766..5786
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5937..5956
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6033..6108
                     /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 ccrggsgggg sggggsgggg sgrggggsgg
      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 gggccggggr 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 tcgccaccat ggtagcaggt catgcctctg gcagccccgc attcgggacc
     1741 gcctctcatt cgaattgcga acatgaagag atccacctcg ccggctcgat ccagccgcat
     1801 ggcgcgcttc tggtcgtcag cgaacatgat catcgcgtca tccaggccag cgccaacgcc
     1861 gcggaatttc tgaatctcgg aagcgtactc ggcgttccgc tcgccgagat cgacggcgat
     1921 ctgttgatca agatcctgcc gcatctcgat cccaccgccg aaggcatgcc ggtcgcggtg
     1981 cgctgccgga tcggcaatcc ctctacggag tactgcggtc tgatgcatcg gcctccggaa
     2041 ggcgggctga tcatcgaact cgaacgtgcc ggcccgtcga tcgatctgtc aggcacgctg
     2101 gcgccggcgc tggagcggat ccgcacggcg ggttcactgc gcgcgctgtg cgatgacacc
     2161 gtgctgctgt ttcagcagtg caccggctac gaccgggtga tggtgtatcg tttcgatgag
     2221 caaggccacg gcctggtatt ctccgagtgc catgtgcctg ggctcgaatc ctatttcggc
     2281 aaccgctatc cgtcgtcgac tgtcccgcag atggcgcggc agctgtacgt gcggcagcgc
     2341 gtccgcgtgc tggtcgacgt cacctatcag ccggtgccgc tggagccgcg gctgtcgccg
     2401 ctgaccgggc gcgatctcga catgtcgggc tgcttcctgc gctcgatgtc gccgtgccat
     2461 ctgcagttcc tgaaggacat gggcgtgcgc gccaccctgg cggtgtcgct ggtggtcggc
     2521 ggcaagctgt ggggcctggt tgtctgtcac cattatctgc cgcgcttcat ccgtttcgag
     2581 ctgcgggcga tctgcaaacg gctcgccgaa aggatcgcga cgcggatcac cgcgcttgag
     2641 agctaagcga cgcgtaaatg attgcagatc cactagttct agagctcgct gatcagcctc
     2701 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2761 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2821 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2881 ttgggaagag aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2941 aagaaccagc tgggctcgag gctagagcgg ccgccactgg ccgtcgtttt acaacgtcgt
     3001 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     3061 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     3121 aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     3181 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     3241 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     3301 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     3361 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     3421 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     3481 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3541 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     3601 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3661 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3721 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3781 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3841 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3901 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3961 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4021 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4081 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4141 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4201 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4261 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4321 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4381 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4441 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4501 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4561 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4621 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4681 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4741 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4801 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4861 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4921 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4981 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5041 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5101 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5161 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5221 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5281 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5341 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5401 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5461 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5521 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     5581 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     5641 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     5701 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgaggcg cgccccccct
     5761 tcaccgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     5821 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     5881 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     5941 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     6001 aaggacgaaa caccgggtct tcgagaagac ctgttttaga gctagaaata gcaagttaaa
     6061 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgatcc
     6121 aagggtgggg cgcgccggat t
//
