LOCUS       sequence-387222-        7745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the fusion protein of Tubulin-miRFP720 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-miRFP720
FEATURES             Location/Qualifiers
     source          1..7745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2052..2070)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2053..2070)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2090..2107)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2096..2320
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2366..2794
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2453..2472)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2663..2684
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2803..2823)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2808..3137
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2988..3123
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3050..3069
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3204..3998
                     /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(3258..3277)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3868..3887
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4172..4293
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4209..4228)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4263..4282
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4342..4358)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4342..4358)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4355..4377)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4366..4382
                     /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(4390..4396,4397..4414,4415..4420))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4435..4456
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4573..4590)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4744..5332)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4824..4843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5503..6294,6295..6363))
                     /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     6126..6145
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6364..6468)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6543..6562)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6734..7113
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7114..7317
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7268..7288
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7362..7381
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7362..7380
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      7400..7409
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7406..7745,1..608)
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 720 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP720
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACKRVAERLATQIGVMEE"
ORIGIN
        1 tcttcctcga gctcgagcct ccccagcggg acgtcgccga gccgcaggcg ttcttccgcc
       61 gcaccaacag cgccatccgc cgcctgcagg ccgccgaaac cttggaaagc gcctgcgccg
      121 ccgcggcgca agaggtgcgg aagattaccg gcttcgatcg ggtgatgatc tatcgcttcg
      181 cctccgactt cagcgggtcc gtgatcgcag aggatcggtg cgccgaggtc gagtcaaaac
      241 taggcctgca ctatcctgcc tcattcatcc cggcgcaggc ccgtcggctc tataccatca
      301 acccggtacg gatcattccc gatatcaatt atcggccggt gccggtcacc ccagacctca
      361 atccggtcac cgggcggccg attgatctta gcttcgccat cctgcgcagc gtctcgccca
      421 accatctgga gttcatgcgc aacataggca tgcacggcac gatgtcgatc tcgattttgc
      481 gcggcgagcg actgtgggga ttgatcgttt gccatcaccg aacgccgtac tacgtcgatc
      541 tcgatggccg ccaagcctgc aagagagtcg ccgagagact ggccacccag atcggcgtga
      601 tggaagaggg aggtggtgga tcaggaggag gtggttctgg aggtggtgga tcaggaggaa
      661 gatctcgagt gcgtgagtgc atctccatcc acgttggcca ggctggtgtc cagattggca
      721 atgcctgctg ggagctctac tgcctggaac acggcatcca gcccgatggc cagatgccaa
      781 gtgacaagac cattggggga ggagatgact ccttcaacac cttcttcagt gagacgggcg
      841 ctggcaagca cgtgccccgg gctgtgtttg tagacttgga acccacagtc attgatgaag
      901 ttcgcactgg cacctaccgc cagctcttcc accctgagca gctcatcaca ggcaaggaag
      961 atgctgccaa taactatgcc cgagggcact acaccattgg caaggagatc attgaccttg
     1021 tgttggaccg aattcgcaag ctggctgacc agtgcaccgg tcttcagggc ttcttggttt
     1081 tccacagctt tggtggggga actggttctg ggttcacctc cctgctcatg gaacgtctct
     1141 cagttgatta tggcaagaag tccaagctgg agttctccat ttacccagca ccccaggttt
     1201 ccacagctgt agttgagccc tacaactcca tcctcaccac ccacaccacc ctggagcact
     1261 ctgattgtgc cttcatggta gacaatgagg ccatctatga catctgtcgt agaaacctcg
     1321 atatcgagcg cccaacctac actaacctta accgccttat tagccagatt gtgtcctcca
     1381 tcactgcttc cctgagattt gatggagccc tgaatgttga cctgacagaa ttccagacca
     1441 acctggtgcc ctacccccgc atccacttcc ctctggccac atatgcccct gtcatctctg
     1501 ctgagaaagc ctaccatgaa cagctttctg tagcagagat caccaatgct tgctttgagc
     1561 cagccaacca gatggtgaaa tgtgaccctc gccatggtaa atacatggct tgctgcctgt
     1621 tgtaccgtgg tgacgtggtt cccaaagatg tcaatgctgc cattgccacc atcaaaacca
     1681 agcgcagcat ccagtttgtg gattggtgcc ccactggctt caaggttggc atcaactacc
     1741 agcctcccac tgtggtgcct ggtggagacc tggccaaggt acagagagct gtgtgcatgc
     1801 tgagcaacac cacagccatt gctgaggcct gggctcgcct ggaccacaag tttgacctga
     1861 tgtatgccaa gcgtgccttt gttcactggt acgtgggtga ggggatggag gaaggcgagt
     1921 tttcagaggc ccgtgaagat atggctgccc ttgagaagga ttatgaggag gttggtgtgg
     1981 attctgttga aggagagggt gaggaagaag gagaggaata ctaaggatcc accggatcta
     2041 gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg
     2101 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2161 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2221 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2281 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2341 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2401 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2461 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2521 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2581 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2641 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2701 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2761 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2821 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2881 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2941 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3001 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3061 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3121 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     3181 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3241 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3301 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3361 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3421 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3481 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3541 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3601 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3661 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3721 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3781 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3841 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3901 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3961 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4021 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4081 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4141 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4201 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4261 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4321 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4381 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4441 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4501 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4561 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4621 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4681 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4741 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4801 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4861 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4921 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4981 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5041 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5101 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5161 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5221 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5281 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5341 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5401 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5461 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5521 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5581 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5641 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5701 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5761 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5821 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5881 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5941 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6001 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6061 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6121 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6181 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6241 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6301 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6361 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6421 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6481 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     6541 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     6601 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     6661 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     6721 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     6781 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     6841 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     6901 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     6961 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     7021 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     7081 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     7141 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     7201 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     7261 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
     7321 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
     7381 gagacccaag cttggtaccg ccaccatggc ggaaggatcc gtcgccaggc agcctgacct
     7441 cttgacctgc gacgatgagc cgatccatat ccccggtgcc atccaaccgc atggactgct
     7501 gctcgccctc gccgccgaca tgacgatcgt tgccggcagc gacaaccttc ccgaactcac
     7561 cggactggcg atcggcgccc tgatcggccg ctctgcggcc gatgtcttcg actcggagac
     7621 gcacaaccgt ctgacgatcg ccttggccga gcccggggcg gccgtcggag caccgatcac
     7681 tgtcggcttc acgatgcgaa aggacgcagg cttcatcggc tcctggcatc gccatgatca
     7741 gctca
//
