LOCUS       sequence-291978-        6138 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 6138)
  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 6138)
  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..6138
                     /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      1688..1697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1694..2641
                     /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(2662..2678)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2662..2678)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2692..2709)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2698..2922
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2972..2989)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2972..2988)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2981..3003)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3129..3584)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3266..3287)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3478..3497
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3611..3715
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3716..3784,3785..4576)
                     /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(3934..3953)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4747..5335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5236..5255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5489..5506
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5623..5644
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5659..5664,5665..5682,5683..5689)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5697..5713
                     /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     5702..5724
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5721..5737
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5721..5737
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5763..6011
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5763..5783
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5934..5953
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6030..6105
                     /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 ttaagtcgcc accatggtag caggtcatgc ctctggcagc cccgcattcg ggaccgcctc
     1741 tcattcgaat tgcgaacatg aagagatcca cctcgccggc tcgatccagc cgcatggcgc
     1801 gcttctggtc gtcagcgaac atgatcatcg cgtcatccag gccagcgcca acgccgcgga
     1861 atttctgaat ctcggaagcg tactcggcgt tccgctcgcc gagatcgacg gcgatctgtt
     1921 gatcaagatc ctgccgcatc tcgatcccac cgccgaaggc atgccggtcg cggtgcgctg
     1981 ccggatcggc aatccctcta cggagtactg cggtctgatg catcggcctc cggaaggcgg
     2041 gctgatcatc gaactcgaac gtgccggccc gtcgatcgat ctgtcaggca cgctggcgcc
     2101 ggcgctggag cggatccgca cggcgggttc actgcgcgcg ctgtgcgatg acaccgtgct
     2161 gctgtttcag cagtgcaccg gctacgaccg ggtgatggtg tatcgtttcg atgagcaagg
     2221 ccacggcctg gtattctccg agtgccatgt gcctgggctc gaatcctatt tcggcaaccg
     2281 ctatccgtcg tcgactgtcc cgcagatggc gcggcagctg tacgtgcggc agcgcgtccg
     2341 cgtgctggtc gacgtcacct atcagccggt gccgctggag ccgcggctgt cgccgctgac
     2401 cgggcgcgat ctcgacatgt cgggctgctt cctgcgctcg atgtcgccgt gccatctgca
     2461 gttcctgaag gacatgggcg tgcgcgccac cctggcggtg tcgctggtgg tcggcggcaa
     2521 gctgtggggc ctggttgtct gtcaccatta tctgccgcgc ttcatccgtt tcgagctgcg
     2581 ggcgatctgc aaacggctcg ccgaaaggat cgcgacgcgg atcaccgcgc ttgagagcta
     2641 agcgacgcgt aaatgattgc agatccacta gttctagagc tcgctgatca gcctcgactg
     2701 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2761 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2821 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2881 aagagaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2941 ccagctgggg ctcgaggcta gagcggccgc cactggccgt cgttttacaa cgtcgtgact
     3001 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     3061 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     3121 gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     3181 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     3241 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     3301 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     3361 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     3421 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     3481 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     3541 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     3601 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3661 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3721 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3781 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3841 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3901 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3961 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4021 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4081 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4141 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4201 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4261 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4321 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4381 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4441 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4501 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4561 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4621 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4681 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4741 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4801 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4861 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4921 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4981 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5041 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5101 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5161 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5221 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5281 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5341 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5401 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5461 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5521 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     5581 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     5641 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     5701 gagcggataa caatttcaca caggaaacag ctatgaccat gaggcgcgcc ccccccttca
     5761 ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     5821 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     5881 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     5941 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     6001 gacgaaacac cgggtcttcg agaagacctg ttttagagct agaaatagca agttaaaata
     6061 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgatccaag
     6121 ggtggggcgc gccggatt
//
