LOCUS       sequence-360140-        4548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CreON knock-in for Halo-beta-actin (amino acid position: before 
            start codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4548)
  AUTHORS   Droogers WJ, Willems J, MacGillavry HD, de Jong APH
  TITLE     Duplex Labeling and Manipulation of Neuronal Proteins Using 
            Sequential CRISPR/Cas9 Gene Editing.
  JOURNAL   eNeuro. 2022 Jul 18;9(4):ENEURO.0056-22.2022. doi: 
            10.1523/ENEURO.0056-22.2022.
  PUBMED    35851300
REFERENCE   2  (bases 1 to 4548)
  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: 
            "10.1523/ENEURO.0056-22"; journalName: "eNeuro"; date: "2022-07-18- 
            18"; volume: "9"; issue: "4"; pages: "ENEURO.0056-22.2022"
COMMENT     Sequence Label: pOC1 Halo-Actb KI
FEATURES             Location/Qualifiers
     source          1..4548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(309..342)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     protein_bind    complement(573..606)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     misc_RNA        639..714
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             865..1755
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIHPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     repeat_region   1849..1989
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1849..1978
                     /label=AAV2 ITR
     rep_origin      2064..2519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2151..2170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2361..2382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2536..2555)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2655..2677
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2715..2733)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2801..2905
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2906..2974,2975..3766)
                     /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(3124..3143)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3937..4525
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4426..4445
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtga tccgacgccg ccatctctag
       61 gcccgcgccg gccccctcgc acagacttgt gggagaagct cggctactcc cctgccccgg
      121 ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga
      181 taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt tctataagag
      241 atacaaatac taaattatta ttttaaaaaa cagcacactc gagaactcac cctaactgta
      301 aagttagtat aacttcgtat agtataaatt atacgaagtt ataagccttg ttttttgcga
      361 tttttgaatt cgttcctcag aggaactgac aagcacccta acatcctatt ggaggctcac
      421 tcacgttttt tctattttgt ttcttgacag cagagctcgt tgctcactgt atagctcagg
      481 ttggcctgac actgatgagg ttctccagtg actgcctcta cctacctact gggatgacag
      541 aggtgtacca ccaagccacg cccgggggat ccataacttc gtatagtata aattatacga
      601 agttataagc cttgtttgtg tgccttgata gttcgccagt tttagagcta gaaatagcaa
      661 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
      721 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca
      781 attctgcaga caaatggctc tagaagcttt cgaagaccct agaccctggc gaactatcaa
      841 ggcacattcg ccatggctag cggaatggca gaaatcggta ctggctttcc attcgacccc
      901 cattatgtgg aagtcctggg cgagcgcatg cactacgtcg atgttggtcc gcgcgatggc
      961 acccctgtgc tgttcctgca cggtaacccg acctcctcct acgtgtggcg caacatcatc
     1021 ccgcatgttg caccgaccca tcgctgcatt gctccagacc tgatcggtat gggcaaatcc
     1081 gacaaaccag acctgggtta tttcttcgac gaccacgtcc gcttcatgga tgccttcatc
     1141 gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg actggggctc cgctctgggt
     1201 ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta ttgcatttat ggagttcatc
     1261 caccctatcc cgacctggga cgaatggcca gaatttgccc gcgagacctt ccaggccttc
     1321 cgcaccaccg acgtcggccg caagctgatc atcgatcaga acgtttttat cgagggtacg
     1381 ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga tggaccatta ccgcgagccg
     1441 ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc caaacgagct gccaatcgcc
     1501 ggtgagccag cgaacatcgt cgcgctggtc gaagaataca tggactggct gcaccagtcc
     1561 cctgtcccga agctgctgtt ctggggcacc ccaggcgttc tgatcccacc ggccgaagcc
     1621 gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg acatcggccc gggtctgaat
     1681 ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga tcgcgcgctg gctgtccacg
     1741 ctcgagattt ccggcagcgc tccctggcga actatcaagg cacactcggg gtcttctcct
     1801 cgaggatcct aggacgcgtt aacttacggt aaatggcccg gcggccgcag gaacccctag
     1861 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     1921 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     1981 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2041 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2101 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     2161 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2221 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     2281 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2341 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     2401 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     2461 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     2521 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     2581 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2641 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     2701 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     2761 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2821 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2881 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2941 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3001 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3061 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3121 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     3181 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3241 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3301 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3361 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3421 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     3481 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3541 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3601 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     3661 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3721 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3781 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3841 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3901 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3961 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4021 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4081 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4141 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4201 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4261 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4321 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4381 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4441 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4501 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gccttttt
//
