LOCUS       sequence-412151-        6663 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses the genetically-encoded fluorescent neurotensin (NTS) 
            sensor GRAB_NTS1.0 in a cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6663)
  AUTHORS   Wang H, Qian T, Zhao Y, Zhuo Y, Wu C, Osakada T, Chen P, Chen Z, Ren
            H, Yan Y, Geng L, Fu S, Mei L, Li G, Wu L, Jiang Y, Qian W, Zhang L,
            Peng W, Xu M, Hu J, Jiang M, Chen L, Tang C, Zhu Y, Lin D, Zhou JN, 
            Li Y
  TITLE     A tool kit of highly selective and sensitive genetically encoded 
            neuropeptide sensors.
  JOURNAL   Science. 2023 Nov 17;382(6672):eabq8173. doi: 
            10.1126/science.abq8173. Epub 2023 Nov 17.
  PUBMED    37972184
REFERENCE   2  (bases 1 to 6663)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2023-11-17"; volume: "382(6672):eabq8173. doi"; pages: " 
            10.1126/science.abq8173. Epub 2023 Nov 17"
COMMENT     Sequence Label: pAAV-EFS-DIO-GRAB_NTS1.0
FEATURES             Location/Qualifiers
     source          1..6663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..80
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   222..351
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        372..583
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    complement(602..635)
                     /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(686..719)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1256..1275
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1515..1536
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1642..1663)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2769..2793)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(2794..2856)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTGD"
     regulatory      2853..2862
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2869..2902
                     /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    2953..2986
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3011..3599
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3064..3084)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3482..3493)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3631..4107
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3830..3849)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4147..4287
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4147..4276
                     /label=AAV2 ITR
     rep_origin      4362..4817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4449..4468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4659..4680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4834..4853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4953..4975
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5013..5031)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5099..5203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5204..5272,5273..6064)
                     /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(5422..5441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6235..6663,1..160)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
       61 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
      121 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
      181 tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc
      241 tcgctcgctc actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
      301 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgca
      361 cgcgtaagct tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
      421 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
      481 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
      541 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca cagtctagag tcgactccgg
      601 aataacttcg tataggatac tttatacgaa gttatgcaga atggtagctg gattgtagct
      661 gctattagca atatgaaacc tcttaataac ttcgtatagc atacattata cgaagttatg
      721 gcgcatatgc tagtacagcg tctcgcgggt ggcattgctg gagagggtgt ggttgctgga
      781 cacgctgtcg gccttcctcg agaaggctgg cctcttcctc ctgcgccgcc acaccgggca
      841 gaggcaggcc agtgtggcca ggaagatgtg gcggaagttg gcagagacga ggttgtacag
      901 gatggggttg atggtggagc tgacgtagaa gagtgcgttg gtcaccatgt agaagtagtg
      961 gtagaagtca tagaggaacg gagtccactg ctcatccgag atgtagcaga acatgaggcg
     1021 ccgcacgtgg tagggcagcc agcagaccac aaaggcgatg accactgcac gtaggacgcg
     1081 cacgccgtgc cgcagggcct ggaccctatt aggctcccgc ccgcgccagc gtgctgctgc
     1141 tccggtgttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     1201 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     1261 gtagttgccg tcgtccttga agaagatggt gcgctcctgg atgtagcctt cgggcatggc
     1321 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc
     1381 gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     1441 gaacttcagg gtcagcttgc cctcggtggc atcgccctca ccctcgccgc ccacgctgaa
     1501 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     1561 ctcctcgccc tttctcacca tgctccctcc ggtaccgccc ttgtacagct cgtccatgcc
     1621 ccaagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt
     1681 ggggtctttc gaaagtttgg actgcacgct caggtagtgg ttgtcgggca gcagcacggg
     1741 gccgtcgccg atgggggtgt tctgctggta gtggtaggcg agctgcacgc cgccgtcctc
     1801 gatgttgtgg cggatattga acgccgcctt gatgccgttc ttctgcttgt cggccttgat
     1861 atagacgttt cctccctcct ccaggtccag cgcgtcggtg tcgcgcggcc cggccggcgc
     1921 gggctcgcca ggggcgccgt tgagctgctc ggccgcctgg cgtaccatga cggtcagctt
     1981 gttggcgatg atggtgttca ggaccgagat gaccaccatg gggaatatga aggacatgaa
     2041 ggtgttgacc tgtatgacga ccttgacggt ggcagtgtgg atggtggggg tgcacaccag
     2101 gccgccggcg tgctggccgt cggcgctgcg gttctgctcg cccatggtga acagcatagg
     2161 caccgccagc agggccgagg cgagccagat ggcgctgatg aacttcttgg tgcggcttcg
     2221 ggacatgagg gtcttggcct tgaaggggtg gcagatggcc aggtagcgct ccacactcag
     2281 gctggccacg ttgagggccg tggcgtaggt gcaggcgtcg cgcaggaagt agtagccgcg
     2341 gcagccggcg tcgccgaagg cccaggggtg gtgcacccag atgaagttgt acagctccac
     2401 gggcatggcc agcagcaggg tgagcaggtc ggacagcgcc aggctgccca ggtggtaatg
     2461 caccgtgctc tgcaggctct gcagcgactt cttccgcgcc agcgtgaacg ccgtcaccgt
     2521 gttgcccacc gtgcccacca cgaagagcgc caggtacacg gcggtcacca gcaccttgga
     2581 gtagatgtcg gtgttcacgt ccagctcgct gctgggtgcc gccaggacgc gctccgacgc
     2641 gttgcccgaa gcgttgccga agcccggggc cagcagcgcc tcctccagtc cggcctgcgc
     2701 ccgctggaag gggtcggcgg ccggcgtgcc cggggttccc ggcgcggagc tgttgaggcg
     2761 accggtggtg ccaacttttt tgtacaaact tgtgtcacca gtggaacctg gaacccagag
     2821 cagcagtacc catagcagga gtgtgtctgt ctccatggtg gcgctagcat aacttcgtat
     2881 aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt attctttaga
     2941 atggtgcaaa gaataacttc gtataatgta tgctatacga agttatgaat tcgatatcaa
     3001 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3061 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3121 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3181 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3241 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3301 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3361 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3421 ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3481 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3541 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     3601 tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg
     3661 cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta
     3721 agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg
     3781 gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa
     3841 ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa
     3901 gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc
     3961 agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc
     4021 aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg
     4081 tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc
     4141 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4201 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4261 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4321 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4381 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     4441 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4501 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4561 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4621 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4681 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     4741 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4801 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4861 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4921 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4981 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5041 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5101 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5161 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5221 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5281 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5341 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5401 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5461 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5521 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5581 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5641 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5701 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5761 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5821 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5881 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5941 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6001 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6061 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6121 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6181 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6241 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6301 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6361 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6421 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6481 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6541 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6601 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6661 ggc
//
