LOCUS       sequence-387221-        7745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the fusion protein of Tubulin-miRFP713 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7745)
  AUTHORS   Zhang H, Papadaki S, Sun X, Wang X, Drobizhev M, Yao L, Rehbock M, 
            Koster RW, Wu L, Namikawa K, Piatkevich KD
  TITLE     Quantitative assessment of near-infrared fluorescent proteins.
  JOURNAL   Nat Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z.
  PUBMED    37666982
REFERENCE   2  (bases 1 to 7745)
  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: "Nat 
            Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z."
COMMENT     Sequence Label: pTubulin-miRFP713
FEATURES             Location/Qualifiers
     source          1..7745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        137..516
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        517..720
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     671..691
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     765..784
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        765..783
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      803..812
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             809..1756
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACKRVAERLATQIGVMEE"
     promoter        complement(3200..3218)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3201..3218)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3238..3255)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3244..3468
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3514..3942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3601..3620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3811..3832
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3951..3971)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3956..4285
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4136..4271
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4198..4217
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4352..5146
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4406..4425)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5016..5035
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5320..5441
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5357..5376)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5411..5430
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5490..5506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5490..5506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5503..5525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5514..5530
                     /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)."
     promoter        complement(join(5538..5544,5545..5562,5563..5568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5583..5604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5721..5738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5892..6480)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5972..5991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6651..7442,7443..7511))
                     /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     7274..7293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7512..7616)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7691..7710)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
       61 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
      121 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
      181 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      241 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      301 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      361 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      421 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      481 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      541 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      601 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      661 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      721 ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat
      781 agggagaccc aagcttggta ccgccaccat ggcggaagga tccgtcgcca ggcagcctga
      841 cctcttgacc tgcgacgatg agccgatcca tatccccggt gccatccaac cgcatggact
      901 gctgctcgcc ctcgccgccg acatgacgat cgttgccggc agcgacaacc ttcccgaact
      961 caccggactg gcgatcggcg ccctgatcgg ccgctctgcg gccgatgtct tcgactcgga
     1021 gacgcacaac cgtctgacga tcgccttggc cgagcccggg gcggccgtcg gagcaccgat
     1081 cactgtcggc ttcacgatgc gaaaggacgc aggcttcatc ggctcctggc atcgccatga
     1141 tcagctcatc ttcctcgagc tcgagcctcc ccagcgggac gtcgccgagc cgcaggcgtt
     1201 cttccgccgc accaacagcg ccatccgccg cctgcaggcc gccgaaacct tggaaagcgc
     1261 ctgcgccgcc gcggcgcaag aggtgcggaa gattaccggc ttcgatcggg tgatgatcta
     1321 tcgcttcgcc tccgacttca gcggcgaagt gatcgcagag gatcggtgcg ccgaggtcga
     1381 gtcaaaacta ggcctgcact atcctgcctc aaccgtgccg gcgcaggccc gtcggctcta
     1441 taccatcaac ccggtacgga tcattcccga tatcaattat cggccggtgc cggtcacccc
     1501 agacctcaat ccggtcaccg ggcggccgat tgatcttagc ttcgccatcc tgcgcagcgt
     1561 ctcgcccgtc catctggaat tcatgcgcaa cataggcatg cacggcacga tgtcgatctc
     1621 gattttgcgc ggcgagcgac tgtggggatt gatcgtttgc catcaccgaa cgccgtacta
     1681 cgtcgatctc gatggccgcc aagcctgcaa gagggtcgcc gagaggctgg ccactcagat
     1741 cggcgtgatg gaagagggag gtggtggatc aggaggaggt ggttctggag gtggtggatc
     1801 aggaggaaga tctcgagtgc gtgagtgcat ctccatccac gttggccagg ctggtgtcca
     1861 gattggcaat gcctgctggg agctctactg cctggaacac ggcatccagc ccgatggcca
     1921 gatgccaagt gacaagacca ttgggggagg agatgactcc ttcaacacct tcttcagtga
     1981 gacgggcgct ggcaagcacg tgccccgggc tgtgtttgta gacttggaac ccacagtcat
     2041 tgatgaagtt cgcactggca cctaccgcca gctcttccac cctgagcagc tcatcacagg
     2101 caaggaagat gctgccaata actatgcccg agggcactac accattggca aggagatcat
     2161 tgaccttgtg ttggaccgaa ttcgcaagct ggctgaccag tgcaccggtc ttcagggctt
     2221 cttggttttc cacagctttg gtgggggaac tggttctggg ttcacctccc tgctcatgga
     2281 acgtctctca gttgattatg gcaagaagtc caagctggag ttctccattt acccagcacc
     2341 ccaggtttcc acagctgtag ttgagcccta caactccatc ctcaccaccc acaccaccct
     2401 ggagcactct gattgtgcct tcatggtaga caatgaggcc atctatgaca tctgtcgtag
     2461 aaacctcgat atcgagcgcc caacctacac taaccttaac cgccttatta gccagattgt
     2521 gtcctccatc actgcttccc tgagatttga tggagccctg aatgttgacc tgacagaatt
     2581 ccagaccaac ctggtgccct acccccgcat ccacttccct ctggccacat atgcccctgt
     2641 catctctgct gagaaagcct accatgaaca gctttctgta gcagagatca ccaatgcttg
     2701 ctttgagcca gccaaccaga tggtgaaatg tgaccctcgc catggtaaat acatggcttg
     2761 ctgcctgttg taccgtggtg acgtggttcc caaagatgtc aatgctgcca ttgccaccat
     2821 caaaaccaag cgcagcatcc agtttgtgga ttggtgcccc actggcttca aggttggcat
     2881 caactaccag cctcccactg tggtgcctgg tggagacctg gccaaggtac agagagctgt
     2941 gtgcatgctg agcaacacca cagccattgc tgaggcctgg gctcgcctgg accacaagtt
     3001 tgacctgatg tatgccaagc gtgcctttgt tcactggtac gtgggtgagg ggatggagga
     3061 aggcgagttt tcagaggccc gtgaagatat ggctgccctt gagaaggatt atgaggaggt
     3121 tggtgtggat tctgttgaag gagagggtga ggaagaagga gaggaatact aaggatccac
     3181 cggatctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct
     3241 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3301 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3361 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3421 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3481 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3541 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3601 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3661 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3721 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3781 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3841 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3901 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     3961 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4021 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4081 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4141 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4201 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     4261 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     4321 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4381 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4441 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4501 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     4561 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4621 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4681 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4741 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4801 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4861 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4921 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4981 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     5041 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     5101 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     5161 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     5221 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5281 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     5341 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5401 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     5461 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5521 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5581 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5641 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5701 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5761 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5821 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5881 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5941 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6001 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6061 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6121 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6181 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6241 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6301 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6361 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6421 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6481 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6541 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6601 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6661 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6721 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6781 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6841 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6901 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6961 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     7021 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7081 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7141 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7201 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7261 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7321 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7381 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7441 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7501 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7561 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7621 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     7681 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     7741 gtgtg
//
