LOCUS       sequence-362419-        6726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template for the stable expression of a mNeonGreen-CAAX 
            fusion in human cells using CRISPR/Cas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6726)
  AUTHORS   Husser MC, Ozugergin I, Resta T, Martin VJJ, Piekny AJ
  TITLE     Cytokinetic diversity in mammalian cells is revealed by the 
            characterization of endogenous anillin, Ect2 and RhoA.
  JOURNAL   Open Biol. 2022 Nov;12(11):220247. doi: 10.1098/rsob.220247. Epub 
            2022 Nov 23.
  PUBMED    36416720
REFERENCE   2  (bases 1 to 6726)
  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: "Open 
            Biol."; date: "2022-11"; volume: "12(11):220247. doi"; pages: " 
            10.1098/rsob.220247. Epub 2022 Nov 23"
COMMENT     Sequence Label: pODN-AAVS1-mNG-CAAX
FEATURES             Location/Qualifiers
     source          1..6726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    880..901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(916..921,922..939,940..946)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    1575..2378
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     enhancer        2430..2733
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2734..2937
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2888..2908
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    2960..3040
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      3042..3051
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3048..3755
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      3303..3312
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3977..4098
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4014..4033)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4068..4087
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    4149..4985
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(5180..5199)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5181..5199)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5505..5523)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5591..5695
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5696..5764,5765..6556)
                     /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(5914..5933)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcaattg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      901 ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg gaattatgag
      961 cggataataa tttcacacag gaggtttaaa ctttaaacat gtcaaaagag acgtcttttg
     1021 ttaagaatgc tgaggaactt gcaaagcaaa aaatggatgc tattaaccct gaactttctt
     1081 caaaatttaa atttttaata aaattcctgt ctcagtttcc tgaagcttgc tctaaacctc
     1141 gttcaaaaaa aatgcagaat aaagttggtc aagaggaaca tattgaatat ttagctcgta
     1201 gttttcatga gagtcgattg ccaagaaaac ccacgccacc tacaacggtt cctgatgagg
     1261 tggttagcat agttcttaat ataagtttta atatacagcc tgaaaatctt gagagaataa
     1321 aagaagaaca tcgattttcc atggcagctg agaatattgt aggagatctt ctagaaagat
     1381 ctcagtctgg tctatctgcc tggccctggc cattgtcact ttgcgctgcc ctcctctcgc
     1441 cccgagtgcc cttgctgtgc cgccggaact ctgccctcta acgctgccgt ctctctcctg
     1501 agtccggacc actttgagct ctactggctt ctgcgccgcc tctggcccac tgcttcccct
     1561 tcccaggcag gtcctgcttt ctctgacctg cattctctcc cctgggcctg tgccgctttc
     1621 tgtctgcagc ttgtggtctg ggtcacctct acggctggcc cagatccttc cctgccgcat
     1681 ccttcaggtt ccgtcttcct ccactccctc ttccccttgc tctctgctgt gttgctgccc
     1741 aaggatgctc tttccggagc acttccttct cggcgctgca ccacgtgatg tcctctgagc
     1801 ggatcctccc cgtgtctggg tcctctccgg gcatctctcc tccctcaccc aaccccatgc
     1861 cgtcttcact cgctgggttc ccttttcctt ctccttctgg ggcctgtgcc atctctcgtt
     1921 tcttaggatg gccttctccg acggatgtct cccttgcgtc ccgcctcccc ttcttgtagg
     1981 cctgcatcat caccgttttt ctggacaacc ccaaagtacc ccgtctccct ggctttagcc
     2041 acctctccat cctcttgctt tctttgcctg gacaccccgt tctcctgtgg attcgggtca
     2101 cctctcaccc ctttcatttg ggcagctccc ctacccccct tacctctcta gtctgtgcta
     2161 gctcttccag ccccctgtca tggcatcttc caggggtccg agagctcagc tagtcttctt
     2221 cctccaaccc gggcccctat gtccacttca ggacagcatg tttgctgcct ccagggatcc
     2281 tgtgtccccg agctgggacc accttatatt cccagggccg gttaatgtgg ctctggttct
     2341 gggtactttt atctgtcccc tcgaccacac agtggggcct agtaatcaat tacggggtca
     2401 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     2461 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     2521 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     2581 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     2641 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     2701 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     2761 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     2821 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     2881 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
     2941 ttagtgaacc gtcagatccg ctagcgctac cggactcaga tctcgagctc aagcttcgaa
     3001 ttctgcagtc gacggtaccg cgggcccggg atccaccggt cgccaccatg gtgagcaagg
     3061 gcgaggagga taacatggcc tctctcccag cgacacatga gttacacatc tttggctcca
     3121 tcaacggtgt ggactttgac atggtgggtc agggcaccgg caatccaaat gatggttatg
     3181 aggagttaaa cctgaagtcc accaagggtg acctccagtt ctccccctgg attctggtcc
     3241 ctcatatcgg gtatggcttc catcagtacc tgccctaccc tgacgggatg tcgcctttcc
     3301 aggccgccat ggtagatggc tccggatacc aagtccatcg cacaatgcag tttgaagatg
     3361 gtgcctccct tactgttaac taccgctaca cctacgaggg aagccacatc aaaggagagg
     3421 cccaggtgaa ggggactggt ttccctgctg acggtcctgt gatgaccaac tcgctgaccg
     3481 ctgcggactg gtgcaggtcg aagaagactt accccaacga caaaaccatc atcagtacct
     3541 ttaagtggag ttacaccact ggaaatggca agcgctaccg gagcactgcg cggaccacct
     3601 acacctttgc caagccaatg gcggctaact atctgaagaa ccagccgatg tacgtgttcc
     3661 gtaagacgga gctcaagcac tccaagaccg agctcaactt caaggagtgg caaaaggcct
     3721 ttaccgatgt gatgggcatg gacgagctgt acaagtccgg actcagatct cgagctcaag
     3781 cttcgaattc aaagatgagc aaagatggta aaaagaagaa aaagaagtca aagacaaagt
     3841 gtgtaattat gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga
     3901 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     3961 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4021 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4081 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     4141 taaattttac tagggacagg cttagtgaca gaaaagcccc atccttaggc ctcctccttc
     4201 ctagtctcct gatattgggt ctaaccccca cctcctgtta ggcagattcc ttatctggtg
     4261 acacaccccc atttcctgga gccatctctc tccttgccag aacctctaag gtttgcttac
     4321 gatggagcca gagaggatcc tgggagggag agcttggcag ggggtgggag ggaagggggg
     4381 gatgcgtgac ctgcccggtt ctcagtggcc accctgcgct accctctccc agaacctgag
     4441 ctgctctgac gcggctgtct ggtgcgtttc actgatcctg gtgctgcagc ttccttacac
     4501 ttcccaagag gagaagcagt ttggaaaaac aaaatcagaa taagttggtc ctgagttcta
     4561 actttggctc ttcacctttc tagtccccaa tttatattgt tcctccgtgc gtcagtttta
     4621 cctgtgagat aaggccagta gccagccccg tcctggcagg gctgtggtga ggaggggggt
     4681 gtccgtgtgg aaaactccct ttgtgagaat ggtgcgtcct aggtgttcac caggtcgtgg
     4741 ccgcctctac tccctttctc tttctccatc cttctttcct taaagagtcc ccagtgctat
     4801 ctgggacata ttcctccgcc cagagcaggg tcccgcttcc ctaaggccct gctctgggct
     4861 tctgggtttg agtccttggc aagcccagga gaggcgctca ggcttccctg tcccccttcc
     4921 tcgtccacca tctcatgccc ctggctctcc tgccccttcc ctacaggggt tcctggctct
     4981 gctcttcaga ctgagccccg ttcccctgca tccccgttcc cctgcatccc ccagaggccc
     5041 caggccacct acttggcctg gaccccacga gaggccaccc cagccctgtc taccaggctg
     5101 ccttttgggt ggattctcct ccaactgtgg ggatcttgct gaaaaactcg agccatccgg
     5161 aagatctggc ggccgctctc cctatagtga gtcgtattac gccggatgga tatggtgttc
     5221 aggcacaagt gttaaagcag ttgattttat tcactatgat gaaaaaaaca atgaatggaa
     5281 cctgctccaa gttaaaaata gagataatac cgaaaactca tcgagtagta agattagaga
     5341 taatacaaca ataaaaaaat ggtttagaac ttactcacag cgtgatgcta ctaattggga
     5401 caattttcca gatgaagtat catctaagaa tttaaatgaa gaagacttca gagcttttgt
     5461 taaaaattat ttggcaaaaa taatataatt cggctgcagg ggcggcctcg tgatacgcct
     5521 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5581 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5641 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5701 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5761 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5821 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5881 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5941 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6001 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6061 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6121 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6181 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6241 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6301 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6361 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6421 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6481 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6541 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6601 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6661 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6721 atcttc
//
