LOCUS       sequence-263461-        6653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bicistronic vector for the expression of EGFP reporter and untagged 
            constitutive active myosin II light chain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6653)
  AUTHORS   Dupraz S, Hilton BJ, Husch A, Santos TE, Coles CH, Stern S, 
            Brakebusch C, Bradke F
  TITLE     RhoA Controls Axon Extension Independent of Specification in the 
            Developing Brain.
  JOURNAL   Curr Biol. 2019 Nov 18;29(22):3874-3886.e9. doi: 
            10.1016/j.cub.2019.09.040. Epub 2019 Oct 31.
  PUBMED    31679934
REFERENCE   2  (bases 1 to 6653)
  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"; doi: 
            "10.1016/j.cub.2019.09.040"; journalName: "Curr Biol"; date: 
            "2019-11-18- 18"; volume: "29"; issue: "22"; pages: "3874-3886.e9"
COMMENT     Sequence Label: pCAG-MLC2(CA)-IRES-GFP
FEATURES             Location/Qualifiers
     source          1..6653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     misc_feature    join(2305..2879,2880..2882,2883..2891)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2472..2489)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2699..2718
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2892..2894,2895..2897,2898..3611)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2892..2894,2895..2897,2898..3608)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2937..2958)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3198..3217)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3545..3566
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3675..3694)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3740..3795
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3794..3813)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4156..4172)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4156..4172)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4169..4191)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4180..4196
                     /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(4204..4210,4211..4228,4229..4234))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4249..4270
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4329..4524
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4375..4510
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4530..4664
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4567..4586)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4621..4640
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4732..4749)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4903..5491)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4983..5002)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5662..6453,6454..6522))
                     /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     6285..6304
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6523..6627)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcctcgaggc
     1741 caccatgtcc agcaagcggg ccaaagccaa gaccaccaag aagcggccac agcgggccga
     1801 cgacaatgtc ttcgcaatgt ttgaccagtc ccagatccag gagtttaagg aagctttcaa
     1861 catgattgac cagaaccgtg atggcttcat tgacaaggag gacctgcacg acatgctggc
     1921 ctcgctgggg aagaacccca cagacgaata cctggagggc atgatgagcg aggccccggg
     1981 gcccatcaac ttcaccatgt tcctcaccat gtttggggag aagctgaacg gcacggaccc
     2041 cgaggatgtg attcgcaacg cctttgcctg cttcgacgag gaagcctcag gtttcatcca
     2101 tgaggaccac ctccgggagc tgctcaccac catgggtgac cgcttcacag atgaggaagt
     2161 ggacgagatg taccgggagg cacccattga taagaaaggc aacttcaact acgtggagtt
     2221 cacccgcatc ctcaaacatg gcgccaagga taaagacgac taggtcgagg atatcgcggc
     2281 cgctacgtaa attcyscsms cccccccctc tccctccccc ccccctaacg ttactggccg
     2341 aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc
     2401 gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag
     2461 gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt
     2521 tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa
     2581 ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc
     2641 aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg
     2701 gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat
     2761 gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa
     2821 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata
     2881 tggccacaac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     2941 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     3001 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     3061 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     3121 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     3181 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     3241 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     3301 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     3361 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     3421 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     3481 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     3541 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     3601 tgtacaagta aagcggccgg ccgccagcac agtggtcgat cgaccaatgc cctggctcac
     3661 aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt
     3721 gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat
     3781 tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat
     3841 gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt
     3901 tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat
     3961 agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct
     4021 ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga
     4081 ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag
     4141 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4201 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4261 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4321 gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4381 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4441 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     4501 tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca
     4561 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4621 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat
     4681 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4741 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4801 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4861 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4921 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4981 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5041 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5101 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5161 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5221 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5281 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5341 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5401 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5461 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5521 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5581 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5641 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5701 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5761 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5821 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5881 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5941 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6001 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6061 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6121 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6181 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6241 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6301 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6361 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6421 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6481 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6541 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6601 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
