LOCUS       sequence-211466-        7728 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 7728)
  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 7728)
  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..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    70..91
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(106..111,112..129,130..136)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    144..160
                     /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     149..171
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     168..184
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     168..184
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     203..223
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        205..223
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        251..477
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             478..658
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    705..830
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1323..1556
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1741..1785
                     /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             3067..3084
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3088..3120
                     /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(3124..3132,3133..3147)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             3175..3231
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     primer_bind     3927..3946
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             4497..4730
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4802..4923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4839..4858)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4893..4912
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4963..5098
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5118..5137)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5119..5137)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5147..5164)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5147..5163)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5156..5178)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5305..5760
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5392..5411)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5602..5623
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5786..5890
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5891..5959,5960..6751)
                     /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(6109..6128)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6922..7510
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7411..7430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7664..7681
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
       61 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
      121 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
      181 tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct
      241 gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt
      301 tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg
      361 tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc
      421 actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg
      481 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      541 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      601 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      661 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      721 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      781 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      841 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
      901 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
      961 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1021 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1081 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1141 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1201 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1261 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1321 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1381 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1441 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1501 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1561 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1621 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1681 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1741 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1801 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1861 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     1921 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     1981 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg
     2041 tatcggttaa cttttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     2101 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaattc
     2161 aaaattctcg acattgatta ttgactagag cttgcatgtc taagctagac ccttcagatt
     2221 aaaaataact gaggtaaggg cctgggtagg ggaggtggtg tgagacgctc ctgtctctcc
     2281 tctatctgcc catcggccct ttggggagga ggaatgtgcc caaggactaa aaaaaggcca
     2341 tggagccaga ggggcgaggg caacagacct ttcatgggca aaccttgggg ccctgctgtc
     2401 tagcatgccc cactacgggt ctaggctgcc catgtaagga ggcaaggcct ggggacaccc
     2461 gagatgcctg gttataatta acccagacat gtggctgccc cccccccccc aacacctgct
     2521 gcctctaaaa ataaccctgt ccctggtgga tcccctgcat gcgaagatct tcgaacaagg
     2581 ctgtggggga ctgagggcag gctgtaacag gcttgggggc cagggcttat acgtgcctgg
     2641 gactcccaaa gtattactgt tccatgttcc cggcgaaggg ccagctgtcc cccgccagct
     2701 agactcagca cttagtttag gaaccagtga gcaagtcagc ccttggggca gcccatacaa
     2761 ggccatgggg ctgggcaagc tgcacgcctg ggtccggggt gggcacggtg cccgggcaac
     2821 gagctgaaag ctcatctgct ctcaggggcc cctccctggg gacagcccct cctggctagt
     2881 cacaccctgt aggctcctct atataaccca ggggcacagg ggctgccctc attctaccac
     2941 cacctccaca gcacagacag acactcagga gccagccagc ctcgaccgga attcgccacc
     3001 atgctgcaga acgagcttgc tcttaagttg gctggacttg atattaacaa gactggagga
     3061 ggttctcatc atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt
     3121 cgggatctgt acgacgatga cgataaggat ctcgcaacaa tggtcgactc atcacgtcgt
     3181 aagtggaata aggcaggtca cgcagtcaga gctataggtc ggctgagctc acccgtggtt
     3241 tccgagcgga tgtaccccga ggacggcgcc ctgaagagcg agatcaagaa ggggctgagg
     3301 ctgaaggacg gcggccacta cgccgccgag gtcaagacca cctacaaggc caagaagccc
     3361 gtgcagctgc ccggcgccta catcgtggac atcaagttgg acatcgtgtc ccacaacgag
     3421 gactacacca tcgtggaaca gtgcgaacgc gccgagggcc gccactccac cggcggcatg
     3481 gacgagctgt acaagggagg tacaggcggg agtctggtga gcaagggcga ggaggataac
     3541 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     3601 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgaggcctt tcagaccgct
     3661 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     3721 ttcatgtacg gctccaaggc ctacattaag cacccagccg acatccccga ctacttcaag
     3781 ctgtccttcc ccgagggctt caggtgggag cgcgtgatga acttcgagga cggcggcatt
     3841 attcacgtga accaggactc ctccctgcag gacggcgtat tcatctacaa ggtgaagctg
     3901 cgcggcacca acttcccccc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     3961 gctacgcgtg acgacctgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta
     4021 tttgacaagg acggggatgg gacgataaca accaaggagc ttgggacggt gttccgatcg
     4081 ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac
     4141 ggtgacggca cattcgactt ccctgagttc ctgacgatga tggcaagaaa aatgaatgac
     4201 acagacagtg aagaggaaat tcgcgaagcg ttccgcgtgt ttgataagga cggcaatggc
     4261 tacatcggcg cagcagagct tcgccacgtg atgacagacc ttggagagaa gttaacagat
     4321 gaggaggttg atgaaatgat cagggtagca gacatcgatg gggatggtca ggtaaactac
     4381 gaagagtttg tacaaatgat gacagcgaag taggaattcg agctcggtac ctttaagacc
     4441 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     4501 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     4561 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     4621 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     4681 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc
     4741 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     4801 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4861 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4921 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     4981 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     5041 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     5101 gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     5161 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     5221 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     5281 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5341 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5401 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5461 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5521 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5581 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5641 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5701 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5761 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5821 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5881 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5941 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     6001 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6061 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6121 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6181 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6241 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6301 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6361 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6421 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6481 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6541 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6601 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6661 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6721 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6781 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6841 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6901 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6961 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7021 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7081 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7141 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7201 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7261 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7321 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7381 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7441 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7501 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7561 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     7621 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     7681 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttg
//
