LOCUS       sequence-484673-        4108 bp    DNA     circular SYN 26-AUG-2025
DEFINITION  For production of template RNA for use in PRINT, GFP under CBh 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4108)
  AUTHORS   Zhang X, Van Treeck B, Horton CA, McIntyre JJR, Palm SM, Shumate JL,
            Collins K
  TITLE     Harnessing eukaryotic retroelement proteins for transgene insertion 
            into human safe-harbor loci.
  JOURNAL   Nat Biotechnol. 2024 Feb 20. doi: 10.1038/s41587-024-02137-y.
  PUBMED    38379101
REFERENCE   2  (bases 1 to 4108)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 26, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2024 Feb 20. doi: 10.1038/s41587-024-02137-y."
COMMENT     Sequence Label: TCARZ-CBh-GFP-SV40PA-GF3-R4A22
FEATURES             Location/Qualifiers
     source          1..4108
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..34
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        15..33
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    220..239
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     enhancer        245..530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        532..811
                     /label=chicken beta-actin promoter
     intron          812..1040
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1052..1061
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1058..1060,1061..1063,1064..1777)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1058..1060,1061..1063,1064..1774)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1103..1124)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1364..1383)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1711..1732
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1784..1918
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1808..1827)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1862..1881
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    1923..1942
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     rep_origin      complement(2353..2941)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2433..2452)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3112..3903,3904..3972))
                     /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     3735..3754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3973..4077)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccaatgatct taagtaatac gactcactat agggagacgg tcaaccgcgt aggagcggtg
       61 accggaattc ggcgggagta actatgactc tcttaaggag tcatagagcc agaacctcct
      121 cgtggtcccg ctgggcacag ggattaattt ttctgtggca aatttgactg gctctagaag
      181 gtcgaccaga tgtccgaggt cgaccagttg tccgtgtgga attgtgagcg ctcacaattc
      241 cacacgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      301 cattgacgtc aatagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      361 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      421 ttgacgtcaa tgacggtaaa tggcccgcct ggcattgtgc ccagtacatg accttatggg
      481 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga
      541 gccccacgtt ctgcttcact ctccccatct ccccccccct ccccaccccc aattttgtat
      601 ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg gggggcgcgc
      661 gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc
      721 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
      781 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgacgctgc cttcgccccg
      841 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
      901 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
      961 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
     1021 ctgaaatcac tttttttcag gttggcgtac ggccaccatg gtgagcaagg gcgaggagct
     1081 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1141 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1201 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1261 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1321 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1381 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1441 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1501 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1561 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1621 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     1681 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1741 cgggatcact ctcggcatgg acgagctgta caagtaaaag cttgatccag acatgataag
     1801 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     1861 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgt
     1921 ggaattgtga gcgctcacaa ttccacagcg gccgctgagg tagataatct ttgtatagtg
     1981 gggggggatc tcatgtaccg ggtttctttt atttgatttt caataaaaca gacggtagct
     2041 aggttcgcaa ggcagccaca agccaaagat aggtagggtg ctcatagtga gtagggacag
     2101 tgccttttga ttcacaacgc gtcaatacca tctgacacgg atacccttac cggacttgtc
     2161 atgatctccc agacttgtcc aaggtggacg ggccaccttt acttaacccg gaaaaggaac
     2221 atatattaat tatatgtgtt cggaaaatag caaaaaaaaa aaaaaaaaaa aaaaagtctt
     2281 catcggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2341 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2401 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     2461 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     2521 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     2581 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     2641 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     2701 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     2761 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     2821 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     2881 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     2941 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3001 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3061 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3121 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3181 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3241 aatgataccg cgagatccac gctcaccggc tccagattta tcagcaataa accagccagc
     3301 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3361 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3421 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3481 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3541 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3601 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     3661 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     3721 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     3781 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     3841 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     3901 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     3961 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4021 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4081 atttccccga aaagtgccac ctgacgtc
//
