LOCUS       sequence-477844-        7054 bp    DNA     circular SYN 07-JUL-2025
DEFINITION  Cre dependent Red fluorescent, negative response-polarity voltage 
            indicator for mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7054)
  TITLE     Schnitzer lab plasmids 2025
REFERENCE   2  (bases 1 to 7054)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pAAV-EF1a-DIO-Varnam2-WPRE
FEATURES             Location/Qualifiers
     source          1..7054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1524..1557)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3023..3032
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3039..3072
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3123..3156
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3181..3769
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3234..3254)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3652..3663)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3801..4277
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4000..4019)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4317..4457
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4317..4446
                     /label=AAV2 ITR
     rep_origin      4532..4987
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4619..4638)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4829..4850
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5004..5023)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5123..5145
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5183..5201)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5269..5373
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5374..5442,5443..6234)
                     /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(5592..5611)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6405..6993
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6894..6913
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgccttaca cctcgttctc gtagcagaac acgttgatgt cgatctggtc cagggggatg
     1621 tactcgccct cgctggtgat cctgctcttc ttatacagct cgtccatccc gccgcccaga
     1681 ttagagtact tggccacggc cacctctctc tgcaccacat atgtctcgtt atcagactcc
     1741 tcgatgcgct ccagcctgtg gtccacggcg tgcactcctg gcatcttgat attgcccact
     1801 gtcttcttgg agcggtatgt ggtcacaaag ttacagtgca ggtgtcctcc tccatccacc
     1861 ttcagggcga tgtcggtata tcccctcagg cctccgtctg cagggtacat catctctgta
     1921 ttgggctccc agcccttggt cttcttctgc atcactgggc cattgctagg gaagttcacg
     1981 cccctcacct tcacgttata caccagctcg ccatcctcca gagaggtgtc ctgtgtcacg
     2041 gtcaccacgc cgccatcctc gtatcttgtc accctctccc aggtaaagcc ctcggggaag
     2101 ctctgcttaa agaaatcggg gatgtcggct ggatacttga tgaaggtcct gctgccgtac
     2161 atgaagctgg tggccaggat gtcaaaggcg aaaggcagtg gtcctccctc gatcactttg
     2221 atcctcattg tctgcacgcc ctcgtatggc ctgccctctc cctctccggt gcacttgaac
     2281 tggtgcccat tgacagaccc ttccatcacg accttcatcc gcatattttc cttaatcagg
     2341 atggacttga agatagtctc atgggcaatg aggcacccca ttccaaacag tggcttagcg
     2401 agcacatcca ggattcccat gagaatggcc tcattgtcga cggacagcac gccactacca
     2461 gacccgaaag cccacacgac aggataaaac acccagagga ttgacaggaa gacgagcaga
     2521 ctgacatagg agctatggta ctcggggctt ttcttcagat cgtctttcca agtggcgtta
     2581 tacatcccgt atgtcaggac tgcctgcatc acacatccag cgataaagta cacccatttg
     2641 aacttgtgtt catcctcgaa ggcgcccaga ataccaaaca cgatcatgaa aatgtctgcg
     2701 ccgatgaccc aagaaatcat cactccgccc atcttggtca tgacgatgag atcgagcagg
     2761 agcagtggtg tggtcagcac ccagctaata tatcttgcgt agaccacctg cctttcacca
     2821 ttcattgtca ggtcctggta gttcacggcc attgcatagt aagcggtagc ggcgatagtg
     2881 cagatggcaa tgttgatata gtatgcgctc ttcagctctg aaggccgaaa gtgcacagcc
     2941 actccaaaag caatagcagc agcagccata atagcaaaga caatccactg tgcaatcatg
     3001 ccggtctcgg tttccacgtc agccatggtg gcgctagcat aacttcgtat aaagtatcct
     3061 atacgaagtt atttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa
     3121 gaataacttc gtataatgta tgctatacga agttatgaat tcgatatcaa gcttatcgat
     3181 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3241 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3301 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3361 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3421 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3481 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3541 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     3601 gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3661 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3721 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga
     3781 gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
     3841 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
     3901 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
     3961 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
     4021 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
     4081 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
     4141 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat
     4201 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     4261 ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga
     4321 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     4381 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4441 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4501 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4561 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     4621 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4681 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4741 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4801 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4861 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     4921 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4981 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     5041 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5101 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5161 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5221 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5281 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5341 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5401 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5461 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5521 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5581 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5641 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5701 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5761 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5821 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5881 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5941 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6001 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6061 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6121 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6181 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6241 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6301 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6361 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6421 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6481 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6541 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6601 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6661 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6721 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6781 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6841 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6901 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6961 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7021 ttcctggcct tttgctggcc ttttgctcac atgt
//
