LOCUS       sequence-456694-        6990 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Membrane tethered mCherry and shRNA targeting Atg7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6990)
  TITLE     Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of 
            ERM Phosphorylation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6990)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pPB-CAG-shAtg7-mCherry-CAAX
FEATURES             Location/Qualifiers
     source          1..6990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     189..208
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(587..606)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    652..707
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(706..725)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   1378..1412
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1435..1451)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1435..1451)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1448..1470)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1459..1475
                     /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(1483..1489,1490..1507,1508..1513))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1528..1549
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1608..1803
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1654..1789
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1809..1943
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1846..1865)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1900..1919
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2011..2028)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2182..2770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2262..2281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2941..3732,3733..3801))
                     /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     3564..3583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3802..3906)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   3944..4006
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    4303..4336
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4497..4745
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4497..4517
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4668..4687
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    4842..4959
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        5059..5362
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5364..5639
                     /label=chicken beta-actin promoter
     intron          5640..6656
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     6580..6602
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     6664..6683
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(6766..6990,1..483)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6911..6929)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
ORIGIN
        1 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
       61 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
      121 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
      181 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
      241 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
      301 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
      361 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
      421 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
      481 aagtgctgca tcatcctgta agcggccgca ctcctcaggt gcaggctgcc tatcagaagg
      541 tggtggctgg tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg
      601 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga
      661 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat
      721 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat
      781 atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga
      841 aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt
      901 tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg
      961 ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc
     1021 tcttatggag atccctcgac ctgcagagct ccagcctaca caatcgctca agacgtgtaa
     1081 tgcttttatt atatattagt cacgatatct ataacaagaa aatatatata taataagtta
     1141 tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat cttaaaagtc
     1201 acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc aaaatcagtg
     1261 acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact gagatgtcct
     1321 aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc aagaatgcat
     1381 gcgtcaattt tacgcagact atctttctag ggttaaaagc ttggcgtaat catggtcata
     1441 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     1501 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     1561 ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaataag
     1621 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     1681 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     1741 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     1801 aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     1861 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     1921 atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg
     1981 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     2041 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     2101 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     2161 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2221 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2281 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2341 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2401 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2461 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2521 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2581 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2641 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2701 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2761 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     2821 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     2881 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2941 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     3001 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     3061 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     3121 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3181 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3241 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3301 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3361 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3421 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3481 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3541 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3601 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3661 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3721 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3781 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     3841 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3901 tccgcgcaca tttccccgaa aagtgccacc tgggtcgact taaccctaga aagataatca
     3961 tattgtgacg tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcgatct
     4021 gtatatcgag gtttatttat taatttgaat agatattaag ttttattata tttacactta
     4081 catactaata ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa
     4141 caaaaactca aaatttcttc tataaagtaa caaaactttt aaacattctc tcttttacaa
     4201 aaataaactt attttgtact ttaaaaacag tcatgttgta ttataaaata agtaattagc
     4261 ttaacttata cataatagaa acaaattata cttattaatc gcataacttc gtataatgta
     4321 tgctatacga agttatgcga ttaagggatc tgtagggcgc agtagtccaa ctagtcaggc
     4381 ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga
     4441 acggatctcg acggtatcga tcacgagact agcctcgagc ggccgccccc ttcaccgagg
     4501 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     4561 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     4621 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     4681 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     4741 acaccggcca gctctgaact caataatact cgagtattat tgagttcaga gctggttttt
     4801 gaattctcga cctcgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     4861 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     4921 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     4981 agagatccac tttggcacta gttattaata gtaatcaatt acggggtcat tagttcatag
     5041 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     5101 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     5161 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     5221 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     5281 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     5341 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
     5401 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
     5461 gatgggggcg gggggggggg gggggcgcgc cagggggggg gggggggggg gagggggggg
     5521 gsggggsgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
     5581 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
     5641 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
     5701 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
     5761 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
     5821 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg
     5881 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct
     5941 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
     6001 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     6061 gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc
     6121 cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg
     6181 gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg
     6241 ggccgcctcg ggccggggrg ggctcggggg aggggcgcgg cggcccccgg agcgccggcg
     6301 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc
     6361 agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc
     6421 cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg
     6481 gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc
     6541 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     6601 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     6661 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc tgcagtcgac
     6721 ggtaccgcgg gcccgctacc ggactcagat ctcgagctca agcttatggt gagcaagggc
     6781 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     6841 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     6901 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     6961 ctgtcccctc agttcatgta cggctccaag
//
