LOCUS       sequence-379173-        6201 bp    DNA     circular SYN 15-APR-2025
DEFINITION  AAV-vector for red fluorescent labeling and green fluorescent 
            calcium measurements from noradrenergic neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6201)
  TITLE     PRSx8-Plasmids
REFERENCE   2  (bases 1 to 6201)
  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"
COMMENT     Sequence Label: PRS_8-jGCaMP7b-P2A-mRuby3
FEATURES             Location/Qualifiers
     source          1..6201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      384..393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(390..398,399..416,417..419,420..452,453..455,456..479,
                     480..506,507..563,564..569,570..839,840..857,858..1292,
                     1293..1298,1299..1739)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNPPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMYARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      486..495
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     776..797
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(903..924)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1164..1183)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1749..1805
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1806..2516
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     misc_feature    2539..3127
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2592..2612)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3010..3021)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3296..3377
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3333..3352)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3387..3406
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3456..3596
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3456..3585
                     /label=AAV2 ITR
     rep_origin      3671..4126
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3758..3777)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3968..3989
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4143..4162)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4262..4284
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4322..4340)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4408..4512
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4513..4581,4582..5373)
                     /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(4731..4750)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5544..6132
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6033..6052
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6194..6201,1..122)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc
       61 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc
      121 ctgcggccgc acgcgtttaa ttaaaaacgc gtattagctt ccgctagaca aatgtgatta
      181 cccccgctag acaaatgtga ttacccgcgc tagacaaatg tgattacccc gctagacaaa
      241 tgtgattacc ccccgctaga caaatgtgat tacccccgct agacaaatgt gattacccgc
      301 gctagacaaa tgtgattacc ccgctagaca aatgtgatta cccccgacca gggcataaat
      361 ggccaggtgg gaccagagat ctggccacca tgggttctca tcatcatcat catcatggta
      421 tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat gacgataagg
      481 atctcgccac catggtcgac tcatcacgtc gtaagtggaa taagacaggt cacgcagtca
      541 gagctatagg tcggctgagc tcactcgaga acgtctatat caaggccgac aagcagaaga
      601 acggcatcaa ggcgaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg
      661 cctaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
      721 actacctgag cgtgcagtcc aaactttcga aagaccccaa cgagaagcgc gatcacatgg
      781 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
      841 gcggtaccgg agggagcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      901 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggtg
      961 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1021 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1081 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacatcc
     1141 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1201 tcgagggcga caccctggtg aaccgcatcg agcttaaggg catcgacttc aaggaggacg
     1261 gcaacatcct ggggcacaag ctggagtaca acccacctga ccaactgact gaagagcaga
     1321 tcgcagaatt taaagagctt ttctccctat ttgacaagga cggggatggg acaataacaa
     1381 ccaaggagct ggggacggtg atgcggtctc tggggcagaa ccccacagaa gcagagctgc
     1441 aggacatgat caatgaagta gatgccgacg gtgacggcac aatcgacttc cctgagttcc
     1501 tgacaatgta cgcaagaaaa atgaaataca gggacacgga agaagaaatt agagaagcgt
     1561 tcggtgtgtt tgataaggat ggcaatggct acatcagtgc agcagagctt cgccacgtga
     1621 tgacaaacct tggagagaag ttaacagatg aagaggttga tgaaatgatc agggaagcag
     1681 acatcgatgg ggatggtcag gtaaactacg aagagtttgt acaaatgatg acagcgaagg
     1741 gatccggagc cacgaacttc tctctgttaa agcaagcagg agacgtggaa gaaaaccccg
     1801 gtcctatggt gtctaagggc gaagagctga tcaaggaaaa tatgcgtatg aaggtggtca
     1861 tggaaggttc ggtcaacggc caccaattca aatgcacagg tgaaggagaa ggcagaccgt
     1921 acgagggaac tcaaaccatg aggatcaaag tcatcgaggg aggacccctg ccatttgcct
     1981 ttgacattct tgccacgtcg ttcatgtatg gcagccgtac ttttatcaag tacccggccg
     2041 acatccctga tttctttaaa cagtcctttc ctgagggttt tacttgggaa agagttacga
     2101 gatacgaaga tggtggagtc gtcaccgtca cgcaggacac cagccttgag gatggcgagc
     2161 tcgtctacaa cgtcaaggtc agaggggtaa actttccctc caatggtccc gtgatgcaga
     2221 agaagaccaa gggttgggag cctaatacag agatgatgta tccagcagat ggtggtctga
     2281 gaggatacac tgacatcgca ctgaaagttg atggtggtgg ccatctgcac tgcaacttcg
     2341 tgacaactta caggtcaaaa aagaccgtcg ggaacatcaa gatgcccggt gtccatgccg
     2401 ttgatcaccg cctggaaagg atcgaggaga gtgacaatga aacctacgta gtgcaacgcg
     2461 aagtggcagt tgccaaatac agcaaccttg gtggtggcat ggacgagctg tacaagtgag
     2521 tttatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     2581 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     2641 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     2701 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     2761 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     2821 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     2881 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     2941 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     3001 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3061 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3121 tccccgcatc gataccgagc gctgctcgag agatctgtga tagcggccat caagctggcc
     3181 gcgactctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     3241 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     3301 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3361 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcagctta
     3421 tcgataccgc atgcacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc
     3481 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     3541 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     3601 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     3661 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3721 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     3781 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3841 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     3901 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3961 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     4021 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     4081 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4141 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4201 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4261 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     4321 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4381 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4441 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4501 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4561 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4621 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4681 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4741 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4801 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4861 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4921 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4981 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5041 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5101 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5161 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5221 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5281 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5341 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5401 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5461 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5521 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5581 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5641 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5701 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5761 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5821 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5881 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5941 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6001 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6061 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6121 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6181 tttgctcaca tgtcctgcag g
//
