LOCUS       sequence-206225-        7714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses calcium indicator R-GECO in muscle cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7714)
  AUTHORS   Rao L, Qian Y, Khodabukus A, Ribar T, Bursac N
  TITLE     Engineering human pluripotent stem cells into a functional skeletal 
            muscle tissue.
  JOURNAL   Nat Commun. 2018 Jan 9;9(1):126. doi: 10.1038/s41467-017-02636-4.
  PUBMED    29317646
REFERENCE   2  (bases 1 to 7714)
  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 
            Commun."; date: "2018-01-9"; volume: "9(1):126. doi"; pages: " 
            10.1038/s41467-017-02636-4"
COMMENT     Sequence Label: pRRL-MHCK7-R-GECO
FEATURES             Location/Qualifiers
     source          1..7714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             236..416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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"
     CDS             2813..2830
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2834..2866
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(2870..2878,2879..2893)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             2921..2977
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     primer_bind     3673..3692
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             4243..4476
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4548..4669
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4585..4604)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4639..4658
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4688..4823
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4855..4874)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4856..4874)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4884..4901)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4884..4900)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4893..4915)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5045..5500
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5132..5151)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5342..5363
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5529..5633
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5634..5702,5703..6494)
                     /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(5852..5871)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6665..7253
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7154..7173
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7407..7424
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7541..7562
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7577..7582,7583..7600,7601..7607)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7615..7631
                     /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     7620..7642
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7639..7655
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7639..7655
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7674..7694
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7676..7694
                     /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 ctcgatacaa taaacgggtc
      241 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      301 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      361 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      421 cgcccgaaca gggacctgaa agcgaaaggg aaaccagagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cctcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggttaac
     1801 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     1861 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattccat
     1921 tgattattga ctagaagctg catgtctaag ctagaccctt cagattaaaa ataactgagg
     1981 taagggcctg ggtaggggag gtggtgtgag acgctcctgt ctctcctcta tctgcccatc
     2041 ggccctttgg ggaggaggaa tgtgcccaag gactaaaaaa aggccatgga gccagagggg
     2101 cgagggcaac agacctttca tgggcaaacc ttggggccct gctgtctagc atgccccact
     2161 acgggtctag gctgcccatg taaggaggca aggcctgggg acacccgaga tgcctggtta
     2221 taattaaccc agacatgtgg ctgccccccc ccccccaaca cctgctgcct ctaaaaataa
     2281 ccctgtccct ggtggatccc ctgcatgcga agatcttcga acaaggctgt gggggactga
     2341 gggcaggctg taacaggctt gggggccagg gcttatacgt gcctgggact cccaaagtat
     2401 tactgttcca tgttcccggc gaagggccag ctgtcccccg ccagctagac tcagcactta
     2461 gtttaggaac cagtgagcaa gtcagccctt ggggcagccc atacaaggcc atggggctgg
     2521 gcaagctgca cgcctgggtc cggggtgggc acggtgcccg ggcaacgagc tgaaagctca
     2581 tctgctctca ggggcccctc cctggggaca gcccctcctg gctagtcaca ccctgtaggc
     2641 tcctctatat aacccagggg cacaggggct gccctcattc taccaccacc tccacagcac
     2701 agacagacac tcaggagcca gccagcctcg accggaattc gccaccatgc tgcagaacga
     2761 gcttgctctt aagttggctg gacttgatat taacaagact ggaggaggtt ctcatcatca
     2821 tcatcatcat ggtatggcta gcatgactgg tggacagcaa atgggtcggg atctgtacga
     2881 cgatgacgat aaggatctcg caacaatggt cgactcatca cgtcgtaagt ggaataaggc
     2941 aggtcacgca gtcagagcta taggtcggct gagctcaccc gtggtttccg agcggatgta
     3001 ccccgaggac ggcgccctga agagcgagat caagaagggg ctgaggctga aggacggcgg
     3061 ccactacgcc gccgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     3121 cgcctacatc gtggacatca agttggacat cgtgtcccac aacgaggact acaccatcgt
     3181 ggaacagtgc gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     3241 gggaggtaca ggcgggagtc tggtgagcaa gggcgaggag gataacatgg ccatcatcaa
     3301 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
     3361 cgagggcgag ggcgagggcc gcccctacga ggcctttcag accgctaagc tgaaggtgac
     3421 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
     3481 caaggcctac attaagcacc cagccgacat ccccgactac ttcaagctgt ccttccccga
     3541 gggcttcagg tgggagcgcg tgatgaactt cgaggacggc ggcattattc acgtgaacca
     3601 ggactcctcc ctgcaggacg gcgtattcat ctacaaggtg aagctgcgcg gcaccaactt
     3661 cccccccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcta cgcgtgacga
     3721 cctgactgaa gagcagatcg cagaatttaa agaggctttc tccctatttg acaaggacgg
     3781 ggatgggacg ataacaacca aggagcttgg gacggtgttc cgatcgctgg ggcagaaccc
     3841 cacagaagca gagctgcagg acatgatcaa tgaagtagat gccgacggtg acggcacatt
     3901 cgacttccct gagttcctga cgatgatggc aagaaaaatg aatgacacag acagtgaaga
     3961 ggaaattcgc gaagcgttcc gcgtgtttga taaggacggc aatggctaca tcggcgcagc
     4021 agagcttcgc cacgtgatga cagaccttgg agagaagtta acagatgagg aggttgatga
     4081 aatgatcagg gtagcagaca tcgatgggga tggtcaggta aactacgaag agtttgtaca
     4141 aatgatgaca gcgaagtagg aattcgagct cggtaccttt aagaccaatg acttacaagg
     4201 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     4261 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     4321 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     4381 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     4441 tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta
     4501 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg
     4561 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4621 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc
     4681 tctagctatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4741 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4801 gtgaggaggc ttttttggag gcctaggctt ttgcgtcgag acgtacccaa ttcgccctat
     4861 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     4921 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     4981 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     5041 cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5101 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     5161 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5221 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     5281 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5341 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5401 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5461 tttaacgcga attttaacaa aatattaacg tttacaattt cccaggtggc acttttcggg
     5521 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5581 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5641 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5701 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5761 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5821 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5881 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5941 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6001 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6061 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6121 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6181 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6241 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6301 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6361 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6421 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6481 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6541 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6601 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6661 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6721 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6781 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     6841 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6901 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6961 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7021 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7081 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7141 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7201 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7261 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7321 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7381 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     7441 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     7501 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     7561 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     7621 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     7681 accctcacta aagggaacaa aagctggagc tgca
//
