LOCUS       sequence-118026-        7088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology Arms and 3X Flag with P2A Neo for 3' tagging of human 
            CREB1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7088)
  AUTHORS   Savic D, Partridge EC, Newberry KM, Smith SB, Meadows SK, Roberts 
            BS, Mackiewicz M, Mendenhall EM, Myers RM
  TITLE     CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins.
  JOURNAL   Genome Res. 2015 Sep 9.
  PUBMED    26355004
REFERENCE   2  (bases 1 to 7088)
  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"; journalName: "Genome Res.
            2015 Sep 9."
COMMENT     Sequence Label: pFETCH_CREB1
FEATURES             Location/Qualifiers
     source          1..7088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      54..642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     543..562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     796..813
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1113..1134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1149..1154,1155..1172,1173..1179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1187..1203
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     1192..1214
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1211..1227
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1211..1227
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1404..1606
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1557..1577
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1603..1627
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1703..2833
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     CDS             4125..4148
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4152..4175
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4179..4202
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4218..4274
                     /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             4287..5081
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4341..4360)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4951..4970
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5712..5729)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5712..5728)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5721..5743)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(5947..6762)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6670..6689
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6754..6773
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgacggagt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
       61 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      121 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      181 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
      241 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      301 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      361 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      421 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      481 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      541 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      601 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      661 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      721 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      781 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagaagctcg cacattcagc
      841 agcgtttttc agcgcgtttt cgatcaacgt ttcaatgttg gtatcaacac caggtttaac
      901 tttgaactta tcggcactga cggttactga ttttgaactt ttgctttgcc acggaacggt
      961 ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cccaatacgc
     1021 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     1081 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     1141 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     1201 caatttcaca caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc
     1261 gactctagag gatccccggg taccgagctc gaattagctt gcatgcctgc aggtcaattc
     1321 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1381 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     1441 agcggtttga ctcacgggga tttcaagtct ccaccccatt gacgtcaatg ggagtttgtt
     1501 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     1561 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     1621 tcagatcgcc tggagacgcc atccacgctg ttttgacctc cataggagac accgggaccg
     1681 atccagcctg gggatcccga ccatggcttc gtacccctgc catcaacacg cgtctgcgtt
     1741 cgaccaggct gcgcgttctc gcggccatag caaccgacgt acggcgttgc gccctcgccg
     1801 gcagcaagaa gccacggaag tccgcctgga gcagaaaatg cccacgctac tgcgggttta
     1861 tatagacggt cctcacggga tggggaaaac caccaccacg caactgctgg tggccctggg
     1921 ttcgcgcgac gatatcgtct acgtacccga gccgatgact tactggcagg tgctgggggc
     1981 ttccgagaca atcgcgaaca tctacaccac acaacaccgc ctcgaccagg gtgagatatc
     2041 ggccggggac gcggcggtgg taatgacaag cgcccagata acaatgggca tgccttatgc
     2101 cgtgaccgac gccgttctgg ctcctcatat cgggggggag gctgggagct cacatgcccc
     2161 gcccccggcc ctcaccctca tcttcgaccg ccatcccatc gccgccctcc tgtgctaccc
     2221 ggccgcgcga taccttatgg gcagcatgac cccccaggcc gtgctggcgt tcgtggccct
     2281 catcccgccg accttgcccg gcacaaacat cgtgttgggg gcccttccgg aggacagaca
     2341 catcgaccgc ctggccaaac gccagcgccc cggcgagcgg cttgacctgg ctatgctggc
     2401 cgcgattcgc cgcgtttacg ggctgcttgc caatacggtg cggtatctgc agggcggcgg
     2461 gtcgtggcgg gaggattggg gacagctttc ggggacggcc gtgccgcccc agggtgccga
     2521 gccccagagc aacgcgggcc cacgacccca tatcggggac acgttattta ccctgtttcg
     2581 ggcccccgag ttgctggccc ccaacggcga cctgtacaac gtgtttgcct gggccttgga
     2641 cgtcttggcc aaacgcctcc gtcccatgca cgtctttatc ctggattacg accaatcgcc
     2701 cgccggctgc cgggacgccc tgctgcaact tacctccggg atggtccaga cccacgtcac
     2761 cacccccggc tccataccga cgatctgcga cctggcgcgc acgtttgccc gggagatggg
     2821 ggaggctaac tgaggatccc acgtcactat tgtatactct atattatact ctatgttata
     2881 ctctgtaatc ctactcaata aacgtgtcac gcctgtgaaa ccgtactaag tctcccgtgt
     2941 attcttatca ccatcaggtg acatcctcgc ccaggctgtc aatcatgccg gtatcgattc
     3001 cagtagcacc ggccccacgc tgacaaccca ctcttgcagc gttagcagcg cccctcttaa
     3061 caagccgacc cccaccagcg tcgcggttac taacactcct ctccccgacc tgcagcccag
     3121 cttccccgac ctgcagccca gcttccccga cctgcagccc agcttccccg acctgcagcc
     3181 cagctaaggt gcggcccaac ctcactcttt tgcatgtgga tatccaggtt tcccagcacc
     3241 atttgttgaa gagactgtta gattttttta aatgccctga aatgattagt ttgtaagatt
     3301 ttctatttaa aggattgtta cacattgaaa tttagcttta atgttttctt agttattgat
     3361 atggatagaa tttcaagttt tgcaggaatt atgcagaata ttttaattaa tagtaagatg
     3421 taatttttaa gtggtcattt aggtcttgtt tgtttatgga gttgtttttt tgtttgtttc
     3481 tttgagacgg agtctcactc ttgtcactca ggctggagtg cagtggcgca atctcagctt
     3541 actgcagcct ctgcctccca ggttcaagcg atgcccatgg ctcagccttc caagtagctg
     3601 ggactacagg catgtgccgc cacaccccgc taatttttgt gtttatagta gagaggggtt
     3661 tcatcatgtt ggccaggctg atcttggact cctgacctca ggtgatctcc ctgccttggc
     3721 ccccgaaagt gctgggatta caggcgtgag ccgccacgcc tggccagatt aaaattttaa
     3781 tataaggtaa tttccaagta attctgtata tcttttccaa tgcttaatat atactaaaat
     3841 gttggttgct gtgagctatt tctttaaaaa agaaaaaaaa aaggtaatcc aaaataaata
     3901 tgtggaaatc atttgcataa tttttcccgt cctcttttgc ttgtagggaa gcagctcgag
     3961 agtgtcgtag aaagaagaaa gaatatgtga aatgtttaga aaacagagtg gcagtgcttg
     4021 aaaatcaaaa caagacattg attgaggagc taaaagcact taaggacctt tactgccaca
     4081 aatcagatgg gagcggagga ggttccggtg gaggtggttc tggagattac aaggatgacg
     4141 acgataaggg cgattacaag gatgacgacg ataagggaga ttacaaggat gacgacgata
     4201 aggtttcagg aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg
     4261 agaaccctgg acctggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     4321 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     4381 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     4441 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     4501 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4561 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4621 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4681 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4741 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4801 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4861 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4921 tgggtgtggc cgaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4981 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     5041 agcgcatcgc cttctatcgc cttcttgacg agttcttctg attcgaacat cgggatttaa
     5101 attttcacct gttaaggtgg aaaatggact ggcttggcca caacctgaaa gacaaaataa
     5161 acattttatt ttctaaacat ttcttttttt ctatgcgcaa aactgcctga aagcaactac
     5221 agaatttcat tcatttgtgc ttttgcatta aactgtgaat gttccaacac ctgcctccac
     5281 ttctcccctc aagaaatttt caacgccagg aatcatgaag agacttctgc ttttcaaccc
     5341 ccaccctcct caagaagtaa taatttgttt acttgtaaat tgatgggaga aatgaggaaa
     5401 agaaaatctt tttaaaaatg atttcaaggt ttgtgctgag ctccttgatt gccttaggga
     5461 cagaattacc ccagcctctt gagctgaagt aatgtgtggg ccgcatgcat aaagtaagta
     5521 aggtgcaatg aagaagtgtt gattgccaaa ttgacatgtt gtcacattct cattgtgaat
     5581 tatgtaaagt tgttaagaga cataccctct aaaaaagaac tttagcatgg tattgaagga
     5641 attagaaatg aatttggagt gctttttatg tcttggaaag tcctctccac tgactgtagc
     5701 ctccaaaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     5761 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     5821 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatgcga tttattcaac
     5881 aaagccgccg tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa
     5941 ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     6001 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     6061 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     6121 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     6181 gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac
     6241 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     6301 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     6361 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     6421 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     6481 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     6541 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     6601 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     6661 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     6721 tcgcggcttc gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     6781 gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta
     6841 acatcagaga ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt
     6901 gaaggatcag atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt
     6961 caaaatcacc aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt
     7021 ctggctggat gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg
     7081 cagacctc
//
