LOCUS       Exported                6500 bp ds-DNA     circular SYN 03-NOV-2021
DEFINITION  Cre-dependent viral expression of the human mu opioid receptor 
            (hMOR/Oprm1) and mCherry. hMOR contains an N-terminal FLAG tag..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Syn1-FLEX-mCh-T2A-FLAG-hMOR-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6500)
  TITLE     Neural basis of opioid-induced respiratory depression and its rescue
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6500)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 3, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6500
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        190..637
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    637..661
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    662..695
                     /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 (Lee and 
                     Saito, 1988)."
     protein_bind    728..761
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1977..2000)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2007..2060)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(2070..2777)
                     /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(2345..2364)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2614..2632
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2774..2783
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(2790..2823)
                     /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 (Lee and 
                     Saito, 1988)."
     protein_bind    complement(2856..2889)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(2890..2914)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2924..3512
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2977..2997)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3395..3406)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3542..3559)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3548..3755
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3763..3903
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3763..3892
                     /label=AAV2 ITR
     rep_origin      3978..4433
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4065..4084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4275..4296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4450..4469)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4569..4591
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4629..4647)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4715..4819
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4820..5680
                     /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(5038..5057)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5851..6439
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6340..6359
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgct gcagagggcc
      181 ctgcgtatga gtgcaagtgg gttttaggac caggatgagg cggggtgggg gtgcctacct
      241 gacgaccgac cccgacccac tggacaagca cccaaccccc attccccaaa ttgcgcatcc
      301 cctatcagag agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc
      361 agcaccgcgg acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac
      421 tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg gccaccttgg
      481 tcgcgtccgc gccgccgccg gcccagccgg accgcaccac gcgaggcgcg agataggggg
      541 gcacgggcgc gaccatctgc gctgcggcgc cggcgactca gcgctgcctc agtctgcggt
      601 gggcagcgga ggagtcgtgt cgtgcctgag agcgcagcaa gtttgtacaa aaaagcaggc
      661 tataacttcg tataggatac tttatacgaa gttatcctcg agtaccggtc acgcgtacgc
      721 aagcttgata acttcgtata gcatacatta tacgaagtta tcctagggcc acctcagggc
      781 aacggagcag tttctgcttc cagattttct agctgatgat tagttctatc cactgtattg
      841 gccgtggagg ggtggtctct agtgttctga cgaattcgag tggagttttg ttgctcaatg
      901 ttggaagagg ttgggataca gaactctctg aagcatcgtt tgaagttttc atccagaaat
      961 gcataaagga ctgggttgag gcagctgttt gtgtaaccta gagcaatgca gaagtgccaa
     1021 gaaacagtct ggaacgtagt ttctgggatt gtaaccaagg ctttaatgat gacgtaaatg
     1081 tgaatgggag tccagcagac gatgaacaca gccaccacca ccagcaccat cctggtgatc
     1141 cttcgaagat tcctgtcctt ttctttggag ccagagagca tgcggacact cttgaggcgc
     1201 aagatcatca gtccatagca cacggtaatg atgagcactg gcataatgaa ggcgaagatg
     1261 aaaacacaga tcttcagcag gttttcccag taccaggttg gatgagagaa tgttagtgta
     1321 caatctatgg aaccttgcct gtattttgtt gtagccatga acattacagg aagaccaatg
     1381 gctgaagaga ggatccagtt gcagacattg ataattttgg catttcgggg agtacggaaa
     1441 tctaaggcct tgacagggtg gcagactgca atgtatcgat caacactcat ggtgcagagg
     1501 gtgaatatgc tggtgaacat gttatagtaa tctatggaga tcactatctt gcaaaggatg
     1561 gttccaaatg gccatgttcc cattaggtaa ttcacactct ggaagggcag ggtactggtg
     1621 gctaaggcat ctgccagagc aaggttgaaa atgtagatgt tggtggcagt cttcatcttg
     1681 gtgtatctga caatcacata catgaccagg aagtttccga agagccccac cacgcacacg
     1741 atggagtaga gggccatgat cgtgatggcc gtgatcatgg agggactgcc ggtcggaggg
     1801 cacaggctgt ctctcccgcc caggtcggtg cggttcggac cgcatgggtc ggacaggttg
     1861 ccatctaagt gggacaagtt gacccaggaa ccggggctgg gtgctgggga gcaacttgag
     1921 tacgccaagg catcagtgca attgctggcg ttcgtggggg cagcgctgct gtccatcttg
     1981 tcgtcatcgt ctttgtaatc tcgcgagggg ccgggatttt cctccacgtc cccgcatgtt
     2041 agaagacttc ccctgccctc tccgcttccc ttgtacagct cgtccatgcc gccggtggag
     2101 tggcggccct cggcgcgttc gtactgttcc acgatggtgt agtcctcgtt gtgggaggtg
     2161 atgtccaact tgatgttgac gttgtaggcg ccgggcagct gcacgggctt cttggccttg
     2221 taggtggtct tgacctcagc gtcgtagtgg ccgccgtcct tcagcttcag cctctgcttg
     2281 atctcgccct tcagggcgcc gtcctcgggg tacatccgct cggaggaggc ctcccagccc
     2341 atggtcttct tctgcattac ggggccgtcg gaggggaagt tggtgccgcg cagcttcacc
     2401 ttgtagatga actcgccgtc ctgcagggag gagtcctggg tcacggtcac cacgccgccg
     2461 tcctcgaagt tcatcacgcg ctcccacttg aagccctcgg ggaaggacag cttcaagtag
     2521 tcggggatgt cggcggggtg cttcacgtag gccttggagc cgtacatgaa ctgaggggac
     2581 aggatgtccc aggcgaaggg cagggggcca cccttggtca ccttcagctt ggcggtctgg
     2641 gtgccctcgt aggggcggcc ctcgccctcg ccctcgatct cgaactcgtg gccgttcacg
     2701 gagccctcca tgtgcacctt gaagcgcatg aactccttga tgatggccat gttatcctcc
     2761 tcgcccttgc tcaccatggt ggctctagaa taacttcgta taaagtatcc tatacgaagt
     2821 tataggtacc tgagctcaac atcgatagca ctagtataac ttcgtataat gtatgctata
     2881 cgaagttata cccagctttc ttgtacaaag tgggaattcc gataatcaac ctctggatta
     2941 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     3001 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     3061 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     3121 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     3181 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     3241 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     3301 cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg
     3361 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     3421 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     3481 gagtcggatc tccctttggg ccgcctcccc gcatcgggaa ttcctagagc tcgctgatca
     3541 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3601 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3661 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3721 gaggattggg aagagaatag caggcatgct ggggagggcc gcaggaaccc ctagtgatgg
     3781 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     3841 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     3901 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     3961 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4021 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     4081 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4141 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     4201 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4261 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     4321 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     4381 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     4441 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4501 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4561 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4621 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4681 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4741 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4801 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4861 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4921 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4981 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5041 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5101 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5161 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5221 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5281 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5341 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5401 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5461 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5521 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5581 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5641 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5701 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5761 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5821 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5881 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5941 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6001 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6061 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6121 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6181 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6241 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6301 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6361 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6421 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6481 ctggcctttt gctcacatgt
//
