LOCUS       sequence-417806-        8270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing the calcium indicator GCaMP6 driven by 
            the muscle-specific promoter MHCK7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8270)
  AUTHORS   Madden L, Juhas M, Kraus WE, Truskey GA, Bursac N
  TITLE     Bioengineered human myobundles mimic clinical responses of skeletal 
            muscle to drugs.
  JOURNAL   eLife. 2015 Jan 9;4:e04885. doi: 10.7554/eLife.04885.
  PUBMED    25575180
REFERENCE   2  (bases 1 to 8270)
  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: "eLife."; 
            date: "2015-01-9"; volume: "4:e04885. doi"; pages: " 
            10.7554/eLife.04885"
COMMENT     Sequence Label: pRRL-MHCK7-GCaMP6
FEATURES             Location/Qualifiers
     source          1..8270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..234
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             235..415
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    462..587
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1080..1313
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1498..1542
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     regulatory      2754..2763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2760..2768,2769..2786,2787..2789,2790..2822,2823..2825,
                     2826..2849,2850..2876,2877..2933,2934..2939,2940..3209,
                     3210..3227,3228..3662,3663..3668,3669..4112)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      2856..2865
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3146..3167
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3273..3294)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3534..3553)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    4121..4709
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4174..4194)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4592..4603)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4796..5029
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5101..5222
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5138..5157)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5192..5211
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5262..5397
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5417..5436)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5418..5436)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5446..5463)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5446..5462)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5455..5477)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5604..6059
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5691..5710)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5901..5922
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6085..6189
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6190..6258,6259..7050)
                     /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(6408..6427)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7221..7809
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7710..7729
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7963..7980
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8097..8118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8133..8138,8139..8156,8157..8163)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8171..8187
                     /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     8176..8198
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8195..8211
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8195..8211
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8230..8250
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8232..8250
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
       61 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      121 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      181 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
      241 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      301 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      361 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
      421 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
      481 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
      541 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
      601 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
      661 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
      721 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
      781 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
      841 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
      901 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
      961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1381 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1681 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     1741 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat
     1801 cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaattcaaa
     1921 attctcgaca ttgattattg actagagctt gcatgtctaa gctagaccct tcagattaaa
     1981 aataactgag gtaagggcct gggtagggga ggtggtgtga gacgctcctg tctctcctct
     2041 atctgcccat cggccctttg gggaggagga atgtgcccaa ggactaaaaa aaggccatgg
     2101 agccagaggg gcgagggcaa cagacctttc atgggcaaac cttggggccc tgctgtctag
     2161 catgccccac tacgggtcta ggctgcccat gtaaggaggc aaggcctggg gacacccgag
     2221 atgcctggtt ataattaacc cagacatgtg gctgcccccc ccccccaaca cctgctgcct
     2281 ctaaaaataa ccctgtccct ggtggatccc ctgcatgcga agatcttcga acaaggctgt
     2341 gggggactga gggcaggctg taacaggctt gggggccagg gcttatacgt gcctgggact
     2401 cccaaagtat tactgttcca tgttcccggc gaagggccag ctgtcccccg ccagctagac
     2461 tcagcactta gtttaggaac cagtgagcaa gtcagccctt ggggcagccc atacaaggcc
     2521 atggggctgg gcaagctgca cgcctgggtc cggggtgggc acggtgcccg ggcaacgagc
     2581 tgaaagctca tctgctctca ggggcccctc cctggggaca gcccctcctg gctagtcaca
     2641 ccctgtaggc tcctctatat aacccagggg cacaggggct gccctcattc taccaccacc
     2701 tccacagcac agacagacac tcaggagcca gccagcctcg accggtcgat ctcgccacca
     2761 tgggttctca tcatcatcat catcatggta tggctagcat gactggtgga cagcaaatgg
     2821 gtcgggatct gtacgacgat gacgataagg atctcgccac catggtcgac tcatcacgtc
     2881 gtaagtggaa taagacaggt cacgcagtca gagctatagg tcggctgagc tcactcgaga
     2941 acgtctatat caaggccgac aagcagaaga acggcatcaa ggcgaacttc aagatccgcc
     3001 acaacatcga ggacggcggc gtgcagctcg cctaccacta ccagcagaac acccccatcg
     3061 gcgacggccc cgtgctgctg cccgacaacc actacctgag cgtgcagtcc aaactttcga
     3121 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     3181 tcactctcgg catggacgag ctgtacaagg gcggtaccgg agggagcatg gtgagcaagg
     3241 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     3301 gccacaagtt cagcgtgtcc ggcgagggtg agggcgatgc cacctacggc aagctgaccc
     3361 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     3421 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     3481 tcaagtccgc catgcccgaa ggctacatcc aggagcgcac catcttcttc aaggacgacg
     3541 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3601 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     3661 acctgccgga ccaactgact gaagagcaga tcgcagaatt taaagaggaa ttctccctat
     3721 ttgacaagga cggggatggg acaataacaa ccaaggagct ggggacggtg atgcggtctc
     3781 tggggcagaa ccccacagaa gcagagctgc aggacatgat caatgaagta gatgccgacg
     3841 gtgacggcac aatcgacttc cctgagttcc tgacaatgat ggcaagaaaa atgaaataca
     3901 gggacacgga agaagaaatt agagaagcgt tcggtgtgtt tgataaggat ggcaatggct
     3961 acatcagtgc agcagagctt cgccacgtga tgacaaacct tggagagaag ttaacagatg
     4021 aagaggttga tgaaatgatc agggaagcag acatcgatgg ggatggtcag gtaaactacg
     4081 aagagtttgt acaaatgatg acagcgaagt gagcgtcgac aatcaacctc tggattacaa
     4141 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     4201 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     4261 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     4321 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     4381 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     4441 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     4501 ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat
     4561 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     4621 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     4681 tcggatctcc ctttgggccg cctccccgcc tggaattcga gctcggtacc tttaagacca
     4741 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     4801 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
     4861 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     4921 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     4981 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
     5041 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     5101 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5161 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5221 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
     5281 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     5341 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     5401 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     5461 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     5521 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     5581 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     5641 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     5701 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5761 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     5821 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5881 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5941 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     6001 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     6061 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     6121 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     6181 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     6241 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     6301 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     6361 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     6421 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     6481 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     6541 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6601 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6661 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6721 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6781 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6841 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6901 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6961 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     7021 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     7081 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     7141 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     7201 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     7261 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     7321 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     7381 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7441 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7501 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7561 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7621 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7681 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7741 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7801 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7861 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7921 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     7981 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     8041 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     8101 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     8161 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     8221 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca
//
