LOCUS       sequence-465758-        6791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector with EF1-alpha promoter and Cre-Out GCaMP6f, enabling to 
            express GCaMP6f in Cre-negative cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6791)
  AUTHORS   Fang YY, Yamaguchi T, Song SC, Tritsch NX, Lin D
  TITLE     A Hypothalamic Midbrain Pathway Essential for Driving Maternal 
            Behaviors.
  JOURNAL   Neuron. 2018 Apr 4;98(1):192-207.e10. doi: 
            10.1016/j.neuron.2018.02.019.
  PUBMED    29621487
REFERENCE   2  (bases 1 to 6791)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.neuron.2018.02"; journalName: "Neuron"; date: "2018-04-4-
            4"; volume: "98"; issue: "1"; pages: "192-207.e10"
COMMENT     Sequence Label: AAV-EF1-alpha-loxP-GCaMP6f-loxP-WPRE
FEATURES             Location/Qualifiers
     source          1..6791
                     /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=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             join(1488..1496,1497..1514,1515..1517,1518..1550,1551..1553,
                     1554..1577,1578..1604,1605..1661,1662..1667,1668..1937,
                     1938..1955,1956..2390,2391..2396,2397..2840)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1584..1593
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1874..1895
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2001..2022)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2262..2281)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(2860..2893)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2918..3506
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2971..2991)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3389..3400)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3538..4014
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3737..3756)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4054..4194
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4054..4183
                     /label=AAV2 ITR
     rep_origin      4269..4724
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4356..4375)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4566..4587
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4741..4760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4860..4882
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4920..4938)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5006..5110
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5111..5179,5180..5971)
                     /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(5329..5348)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6142..6730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6631..6650
                     /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 tagcatacat tatacgaagt tatagtggct agacaccatg ggttctcatc
     1501 atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt cgggatctgt
     1561 acgacgatga cgataaggat ctcgccacca tggtcgactc atcacgtcgt aagtggaata
     1621 agacaggtca cgcagtcaga gctataggtc ggctgagctc actcgagaac gtctatatca
     1681 aggccgacaa gcagaagaac ggcatcaagg cgaacttcaa gatccgccac aacatcgagg
     1741 acggcggcgt gcagctcgcc taccactacc agcagaacac ccccatcggc gacggccccg
     1801 tgctgctgcc cgacaaccac tacctgagcg tgcagtccaa actttcgaaa gaccccaacg
     1861 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1921 tggacgagct gtacaagggc ggtaccggag ggagcatggt gagcaagggc gaggagctgt
     1981 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     2041 gcgtgtccgg cgagggtgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     2101 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     2161 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     2221 tgcccgaagg ctacatccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     2281 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2341 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac ctgccggacc
     2401 aactgactga agagcagatc gcagaattta aagaggaatt ctccctattt gacaaggacg
     2461 gggatgggac aataacaacc aaggagctgg ggacggtgat gcggtctctg gggcagaacc
     2521 ccacagaagc agagctgcag gacatgatca atgaagtaga tgccgacggt gacggcacaa
     2581 tcgacttccc tgagttcctg acaatgatgg caagaaaaat gaaatacagg gacacggaag
     2641 aagaaattag agaagcgttc ggtgtgtttg ataaggatgg caatggctac atcagtgcag
     2701 cagagcttcg ccacgtgatg acaaaccttg gagagaagtt aacagatgaa gaggttgatg
     2761 aaatgatcag ggaagcagac atcgatgggg atggtcaggt aaactacgaa gagtttgtac
     2821 aaatgatgac agcgaagtaa ggcgcgccaa cctcgagcca taacttcgta tagcatacat
     2881 tatacgaagt tatgaattcg atatcaagct tatcgataat caacctctgg attacaaaat
     2941 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3001 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3061 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     3121 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3181 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3241 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3301 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct
     3361 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3421 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     3481 gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctacggg
     3541 tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg
     3601 cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc
     3661 tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc
     3721 tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc
     3781 actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg
     3841 ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt
     3901 ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg
     3961 cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt
     4021 ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     4081 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4141 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     4201 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     4261 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4321 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     4381 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4441 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     4501 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4561 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     4621 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     4681 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     4741 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4801 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4861 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     4921 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     4981 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5041 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5101 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5161 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5221 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5281 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5341 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5401 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5461 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5521 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5581 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5641 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5701 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5761 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5821 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5881 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5941 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6001 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6061 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6121 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6181 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6241 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6301 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6361 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6421 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6481 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6541 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6601 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6661 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6721 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6781 tgctcacatg t
//
