LOCUS       sequence-161646-        4539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For insertion of gRNA array containing 11-30 gRNA modules.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4539)
  AUTHORS   Vad-Nielsen J, Lin L, Bolund L, Nielsen AL, Luo Y
  TITLE     Golden Gate Assembly of CRISPR gRNA expression array for 
            simultaneously targeting multiple genes.
  JOURNAL   Cell Mol Life Sci. 2016 May 13.
  PUBMED    27178736
REFERENCE   2  (bases 1 to 4539)
  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: "Cell Mol 
            Life Sci. 2016 May 13."
COMMENT     Sequence Label: pMA-MsgRNA-EGFP
FEATURES             Location/Qualifiers
     source          1..4539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        470..773
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        774..977
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     928..948
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    1018..1074
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1078..1087
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1084..1086,1087..1089,1090..1803)
                     /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(1084..1086,1087..1089,1090..1800)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1129..1150)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1390..1409)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1737..1758
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1926..2047
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1963..1982)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2017..2036
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(2045..2368)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGY
                     STP"
     primer_bind     2233..2249
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2259..2277
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2325..2345)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2325..2343)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2364..2380)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2377..2399)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2388..2404
                     /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(2412..2418,2419..2436,2437..2442))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2457..2478
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2682..2699)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2792..3380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2872..2891)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3551..4342,4343..4411))
                     /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     4174..4193
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4412..4516)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca
      361 aggccacgtg tcttgtccag agctcggatc cgaattggcc tccacggcct agttattaat
      421 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      481 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      541 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      601 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      661 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      721 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      781 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      841 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      901 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      961 tctatataag cagagctggt ttagtgaacc gtcagatccg ctagcgctac cggactcaga
     1021 tctcgagctc aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg atccatcgcc
     1081 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1141 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1201 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1261 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1321 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1381 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1441 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1501 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1561 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     1621 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1681 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1741 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1801 taaagcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     1861 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     1921 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     1981 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2041 tatcttaagg cgtactatat ccctggagac gggcgccgct acagggcgcg tcccattcgc
     2101 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     2161 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     2221 agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg
     2281 aattgggtac cgggcccccc ctcgaggtcc tccagctttt gttcccttta gtgagggtta
     2341 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2401 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     2461 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccaccg gtcgtctccc
     2521 tattggcctc cacggccttg tacaacgcgt ggtacctgga gcacaagact ggcctcacgg
     2581 gccttccgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaacat
     2641 ggtcatagct gtttccttgc gtattgggcg ctctccgctt cctcgctcac tgactcgctg
     2701 cgctcggtcg ttcgggtaaa gcctggggtg cctaatgagc aaaaggccag caaaaggcca
     2761 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2821 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2881 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2941 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3001 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3061 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3121 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3181 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3241 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3301 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3361 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3421 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3481 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3541 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3601 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3661 catctggccc cagtgctgca atgataccgc gagaaccacg ctcaccggct ccagatttat
     3721 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3781 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3841 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3901 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3961 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4021 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4081 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4141 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4201 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4261 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4321 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4381 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4441 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4501 aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
