LOCUS       sequence-387219-        7730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the fusion protein of Tubulin-miRFP670-2 in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7730)
  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 7730)
  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-miRFP670-2
FEATURES             Location/Qualifiers
     source          1..7730
                     /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..1741
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAKRFAERLSTHFTAAHHQR"
     promoter        complement(3185..3203)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3186..3203)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3223..3240)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3229..3453
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3499..3927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3586..3605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3796..3817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3936..3956)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3941..4270
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4121..4256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4183..4202
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4337..5131
                     /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(4391..4410)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5001..5020
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5305..5426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5342..5361)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5396..5415
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5475..5491)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5475..5491)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5488..5510)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5499..5515
                     /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(5523..5529,5530..5547,5548..5553))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5568..5589
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5706..5723)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5877..6465)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5957..5976)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6636..7427,7428..7496))
                     /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     7259..7278
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7497..7601)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7676..7695)
                     /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 ggcgcgtaag gtcgatctca cctcctgcga
      841 tcgcgagccg atccacatcc ccggcagcat tcagccgtgc ggctgcctgc tagcctgcga
      901 cgcgcaggcg gtgcggatca cgcgcattac ggaaaatgcc ggcgcgttct ttggacgcga
      961 aactccgcgg gtcggtgagc tactcgccga ttacttcggc gagaccgaag cccatgcgct
     1021 gcgcaacgca ctggcgcagt cctccgatcc aaagcgaccg gcgctgatct tcggttggcg
     1081 cgacggcctg accggccgca ccttcgacat ctcactgcat cgccatgacg gtacatcgat
     1141 catcgagttc gagcctgcgg cggccgaaca ggccgacaat ccgctgcggc tgacgcggca
     1201 gatcatcgcg cgcaccaaag aactgaagtc gctcgaagag atggccgcac gggtgccgcg
     1261 ctatctgcag gcgatgctcg gctatcaccg cgtgatgttg taccgcttcg cggacgacgg
     1321 ctccgggatg gtgatcggcg aggcgaagcg cagcgacctc gagagctttc tcggtcagca
     1381 ctttccggcg tcgctggtcc cgcagcaggc gcggctactg tacttgaaga acgcgatccg
     1441 cgtggtctcg gattcgcgcg gcatcagcag ccggatcgtg cccgagcacg acgcctccgg
     1501 cgccgcgctc gatctgtcgt tcgcgcacct gcgcagcatc tcgccctgcc atctcgaatt
     1561 tctgcggaac atgggcgtca gcgcctcgat gtcgctgtcg atcatcattg acggcacgct
     1621 atggggattg atcatctgtc atcattacga gccgcgtgcc gtgccgatgg cgcagcgcgt
     1681 cgcggccaaa aggttcgccg agcgcttatc gacgcacttc accgccgccc accaccaacg
     1741 cggaggtggt ggatcaggag gaggtggttc tggaggtggt ggatcaggag gaagatctcg
     1801 agtgcgtgag tgcatctcca tccacgttgg ccaggctggt gtccagattg gcaatgcctg
     1861 ctgggagctc tactgcctgg aacacggcat ccagcccgat ggccagatgc caagtgacaa
     1921 gaccattggg ggaggagatg actccttcaa caccttcttc agtgagacgg gcgctggcaa
     1981 gcacgtgccc cgggctgtgt ttgtagactt ggaacccaca gtcattgatg aagttcgcac
     2041 tggcacctac cgccagctct tccaccctga gcagctcatc acaggcaagg aagatgctgc
     2101 caataactat gcccgagggc actacaccat tggcaaggag atcattgacc ttgtgttgga
     2161 ccgaattcgc aagctggctg accagtgcac cggtcttcag ggcttcttgg ttttccacag
     2221 ctttggtggg ggaactggtt ctgggttcac ctccctgctc atggaacgtc tctcagttga
     2281 ttatggcaag aagtccaagc tggagttctc catttaccca gcaccccagg tttccacagc
     2341 tgtagttgag ccctacaact ccatcctcac cacccacacc accctggagc actctgattg
     2401 tgccttcatg gtagacaatg aggccatcta tgacatctgt cgtagaaacc tcgatatcga
     2461 gcgcccaacc tacactaacc ttaaccgcct tattagccag attgtgtcct ccatcactgc
     2521 ttccctgaga tttgatggag ccctgaatgt tgacctgaca gaattccaga ccaacctggt
     2581 gccctacccc cgcatccact tccctctggc cacatatgcc cctgtcatct ctgctgagaa
     2641 agcctaccat gaacagcttt ctgtagcaga gatcaccaat gcttgctttg agccagccaa
     2701 ccagatggtg aaatgtgacc ctcgccatgg taaatacatg gcttgctgcc tgttgtaccg
     2761 tggtgacgtg gttcccaaag atgtcaatgc tgccattgcc accatcaaaa ccaagcgcag
     2821 catccagttt gtggattggt gccccactgg cttcaaggtt ggcatcaact accagcctcc
     2881 cactgtggtg cctggtggag acctggccaa ggtacagaga gctgtgtgca tgctgagcaa
     2941 caccacagcc attgctgagg cctgggctcg cctggaccac aagtttgacc tgatgtatgc
     3001 caagcgtgcc tttgttcact ggtacgtggg tgaggggatg gaggaaggcg agttttcaga
     3061 ggcccgtgaa gatatggctg cccttgagaa ggattatgag gaggttggtg tggattctgt
     3121 tgaaggagag ggtgaggaag aaggagagga atactaagga tccaccggat ctagagggcc
     3181 ctattctata gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta
     3241 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3301 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3361 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3421 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3481 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3541 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3601 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3661 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3721 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3781 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3841 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3901 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     3961 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4021 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4081 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4141 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4201 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4261 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     4321 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4381 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4441 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4501 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4561 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4621 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4681 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4741 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4801 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4861 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4921 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4981 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5041 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5101 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5161 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5221 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5281 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     5341 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5401 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     5461 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5521 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5581 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5641 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5701 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5761 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5821 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5881 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5941 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6001 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6061 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6121 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6181 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6241 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6301 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6361 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6421 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6481 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6541 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6601 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6661 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6721 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6781 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6841 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6901 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6961 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7021 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7081 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7141 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7201 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7261 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7321 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7381 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7441 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7501 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7561 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7621 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     7681 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
//
