LOCUS       Exported                6980 bp ds-DNA     circular SYN 04-JUL-2024
DEFINITION  Express Rpl22HA in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6980)
  AUTHORS   Baljit Khakh
  TITLE     RiboTag AAV
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6980)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jul 5, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6980
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     128..145
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    262..283
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        298..328
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    336..352
                     /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     341..363
                     /label=No.55 M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     360..376
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     360..376
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   414..543
                     /label=AAV2 ITR
     primer_bind     complement(425..436)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(426..441)
                     /label=No.37_Addgene-ITR_AAV2_Rv
     primer_bind     481..496
                     /label=No.37_Addgene-ITR_AAV2_Rv
     primer_bind     486..497
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(522..542)
                     /label=No.36_Addgene-ITR_AAV2_Fw
     primer_bind     523..543
                     /label=No.35_ITR-primer-Fw&Rv
     misc_feature    604..1284
                     /label=GfaABC1D promoter
     primer_bind     complement(949..960)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     1202..1222
                     /label=No56. GfaABC1D promotor
     intron          1394..1526
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1509..1528
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1571..1589
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1590..1606
                     /label=EGFP-5'insert
     primer_bind     1671..1685
                     /label=EGFP-5'insert
     protein_bind    complement(1698..1731)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
     primer_bind     1742..1762
                     /label=No.47_5-loxp
     protein_bind    complement(1794..1827)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     1836..1868
                     /label=No.52 mNeonGreen-Rv
     CDS             complement(1851..2561)
                     /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      2297..2306
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     complement(2556..2568)
                     /label=No.49 mScarlet-I-Fw
     regulatory      2558..2567
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    2584..2617
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
     primer_bind     complement(2635..2655)
                     /label=No.48_3-lop
     protein_bind    2688..2721
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(2739..2768)
                     /label=No1.EGFP-3'insert
     misc_feature    2762..2786
                     /label=JN011-mini cloning site
     primer_bind     2772..2804
                     /label=No.49 mScarlet-I-Fw
     regulatory      2793..2802
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2799..3494
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2932..2950)
                     /label=No57. mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3200..3219
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3425..3461)
                     /label=No.49 mScarlet-I-Fw
     primer_bind     complement(3480..3495)
                     /label=No.52 mNeonGreen-Rv
     primer_bind     3485..3554
                     /label=No.41_WPRE-Fw-1
     primer_bind     complement(3516..3548)
                     /label=No.50 mScarlet-I-Rv
     misc_feature    3535..4142
                     /label=WPRE fron XCaMP-R
     misc_feature    3538..4130
                     /label=WPRE
                     /note="/vntifkey=21"
     primer_bind     complement(3549..3569)
                     /label=No24.WPRE-sec check
     primer_bind     complement(4125..4157)
                     /label=No.42_WPRE-Rv-1
     primer_bind     complement(4142..4157)
                     /label=No.43_WPRE-Rv-2
     polyA_signal    4160..4281
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4160..4177)
                     /label=No.43_WPRE-Rv-2
     primer_bind     complement(4171..4190)
                     /label=No.46_SV40-Rv
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4225..4244
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4443..4572
                     /label=AAV2 ITR
     primer_bind     complement(4443..4463)
                     /label=No.35_ITR-primer-Fw&Rv
     primer_bind     4444..4464
                     /label=No.36_Addgene-ITR_AAV2_Fw
     primer_bind     complement(4489..4500)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4490..4505)
                     /label=No.37_Addgene-ITR_AAV2_Rv
     primer_bind     4545..4560
                     /label=No.37_Addgene-ITR_AAV2_Rv
     primer_bind     4550..4561
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4591..4608)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4591..4607)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4600..4622)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4749..5204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4836..4855)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5046..5067
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5230..5334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5335..6195
                     /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"
     misc_feature    5538..5548
                     /label=1-sechceck-egfp
     primer_bind     complement(5553..5572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6366..6954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6855..6874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
       61 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
      121 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
      181 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
      241 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
      301 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
      361 aggaaacagc tatgaccatg attacgccag atttaattaa ggccttaatt aggctgcgcg
      421 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
      481 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
      541 ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga
      601 tctaacatat cctggtgtgg agtaggggac gctgctctga cagaggctcg ggggcctgag
      661 ctggctctgt gagctgggga ggaggcagac agccaggcct tgtctgcaag cagacctggc
      721 agcattgggc tggccgcccc ccagggcctc ctcttcatgc ccagtgaatg actcaccttg
      781 gcacagacac aatgttcggg gtgggcacag tgcctgcttc ccgccgcacc ccagcccccc
      841 tcaaatgcct tccgagaagc ccattgagca gggggcttgc attgcacccc agcctgacag
      901 cctggcatct tgggataaaa gcagcacagc cccctagggg ctgcccttgc tgtgtggcgc
      961 caccggcggt ggagaacaag gctctattca gcctgtgccc aggaaagggg atcaggggat
     1021 gcccaggcat ggacagtggg tggcaggggg ggagaggagg gctgtctgct tcccagaagt
     1081 ccaaggacac aaatgggtga ggggagagct ctccccatag ctgggctgcg gcccaacccc
     1141 accccctcag gctatgccag ggggtgttgc caggggcacc cgggcatcgc cagtctagcc
     1201 cactccttca taaagccctc gcatcccagg agcgagcaga gccagagcag gttggagagg
     1261 agacgcatca cctccgctgc tcgcaagctt tattgcggta gtttatcaca gttaaattgc
     1321 taacgcagtc agtgcttctg acacaacagt ctcgaactta agctgcagaa gttggtcgtg
     1381 aggcactggg caggtaagta tcaaggttac aagacaggtt taaggagacc aatagaaact
     1441 gggcttgtcg agacagagaa gactcttgcg tttctgatag gcacctattg gtcttactga
     1501 catccacttt gcctttctct ccacaggtgt ccactcccag ttcaattaca gctcttaagg
     1561 ctagagtact taatacgact cactataggc tagagcctct gctaaccatg ttcatgcctt
     1621 cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg
     1681 caaagaattg gatccccata acttcgtata aagtatccta tacgaagtta tatcaaaata
     1741 ggaagaccaa tgcttcacca tcgacccgaa ttgccaagca tcaccatcga cccataactt
     1801 cgtataatgt atgctatacg aagttatact agggatcccc cctcgagttt tcacttgtac
     1861 agctcgtcca tgcccatcac atcggtaaag gccttttgcc actccttgaa gttgagctcg
     1921 gtcttggagt gcttgagctc cgtcttacgg aacacgtaca tcggctggtt cttcagatag
     1981 ttagccgcca ttggcttggc aaaggtgtag gtggtccgcg cagtgctccg gtagcgcttg
     2041 ccatttccag tggtgtaact ccacttaaag gtactgatga tggttttgtc gttggggtaa
     2101 gtcttcttcg acctgcacca gtccgcagcg gtcagcgagt tggtcatcac aggaccgtca
     2161 gcagggaaac cagtcccctt cacctgggcc tctcctttga tgtggcttcc ctcgtaggtg
     2221 tagcggtagt taacagtaag ggaggcacca tcttcaaact gcattgtgcg atggacttgg
     2281 tatccggagc catctaccat ggcggcctgg aaaggcgaca tcccgtcagg gtagggcagg
     2341 tactgatgga agccataccc gatatgaggg accagaatcc agggggagaa ctggaggtca
     2401 cccttggtgg acttcaggtt taactcctca taaccatcat ttggattgcc ggtgccctga
     2461 cccaccatgt caaagtccac accgttgatg gagccaaaga tgtgtaactc atgtgtcgct
     2521 gggagagagg ccatgttatc ctcctcgccc ttgctcacca tggtggcggt accgaattct
     2581 agtataactt cgtataggat actttatacg aagttatcat tgggattctt cctattttga
     2641 tccaagcatc accatcgacc ctctagtcca gatctcacca tcgacccata acttcgtata
     2701 gcatacatta tacgaagtta tgtccctcga agaggttcga attcgatatc aagcttctgc
     2761 agtcgacgct agcgggccca ctagtaccgg tcgccaccat ggtgagcaag ggcgaggcag
     2821 tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac ggccacgagt
     2881 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     2941 aggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct cagttcatgt
     3001 acggctccag ggccttcatc aagcaccccg ccgacatccc cgactactat aagcagtcct
     3061 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gccgtgaccg
     3121 tgacccagga cacctccctg gaggacggca ccctgatcta caaggtgaag ctccgcggca
     3181 ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg gaagcgtcca
     3241 ccgagcggtt gtaccccgag gacggcgtgc tgaagggcga cattaagatg gccctgcgcc
     3301 tgaaggacgg cggccgctac ctggcggact tcaagaccac ctacaaggcc aagaagcccg
     3361 tgcagatgcc cggcgcctac aacgtcgacc gcaagttgga catcacctcc cacaacgagg
     3421 actacaccgt ggtggaacag tacgaacgct ccgagggccg ccactccacc ggcggcatgg
     3481 acgagctgta caagtaaagc ggccgcgact ctagatcata atcagccata ccacgcataa
     3541 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3601 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3661 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3721 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3781 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3841 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3901 gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc
     3961 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     4021 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     4081 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc ggccggccgc
     4141 ttgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     4201 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     4261 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     4321 ggggagatgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat
     4381 aaggatcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta
     4441 caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4501 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4561 gcgagcgcgc agccttaatt aacctaattc actggccgtc gttttacaac gtcgtgactg
     4621 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     4681 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     4741 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4801 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4861 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4921 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4981 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5041 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5101 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5161 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5221 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5281 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5341 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5401 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5461 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5521 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5581 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5641 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5701 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5761 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5821 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5881 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5941 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6001 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6061 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6121 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6181 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6241 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6301 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6361 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6421 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6481 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6541 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6601 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6661 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6721 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6781 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6841 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6901 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6961 agcaacgcgg cctttttacg
//
