LOCUS       sequence-439427-        6928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Edits NPAS2 Gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6928)
  AUTHORS   Crawford KD, Khan AG, Lopez SC, Goodarzi H, Shipman SL
  TITLE     High throughput variant libraries and machine learning yield design 
            rules for retron gene editors.
  JOURNAL   Nucleic Acids Res. 2025 Jan 11;53(2):gkae1199. doi: 
            10.1093/nar/gkae1199.
  PUBMED    39658047
REFERENCE   2  (bases 1 to 6928)
  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: "Nucleic 
            Acids Res."; date: "2025-01-11"; volume: "53(2):gkae1199. doi"; 
            pages: " 10.1093/nar/gkae1199"
COMMENT     Sequence Label: pAGK43
FEATURES             Location/Qualifiers
     source          1..6928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        368..645
                     /label=chicken beta-actin promoter
     intron          646..1662
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1586..1608
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1670..1689
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1733..1742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1739..1741,1742..1744,1745..2455)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1739..1741,1742..1744,1745..2455)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1784..1805)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2045..2064)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2392..2413
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2465..2521
                     /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             3485..3505
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3533..3757
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3792..4006
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3927..3946
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(4413..4429)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4413..4429)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4426..4448)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4437..4453
                     /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)."
     promoter        complement(join(4461..4467,4468..4485,4486..4491))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4506..4527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4586..4781
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4632..4767
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4787..4921
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4824..4843)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4878..4897
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4989..5006)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5160..5748)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5240..5259)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5919..6710,6711..6779))
                     /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     6542..6561
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6780..6884)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6915..6928,1..366)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
      421 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
      481 gggggggggs gcgcgccrgg sggggsgggg gggggsgggg ggsggggsgg ggsgrggcgg
      541 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
      601 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      661 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      721 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      781 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
      841 cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg
      901 gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
      961 gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg
     1021 tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga
     1081 gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt
     1141 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
     1201 gtgccgggcg gggggtggcg gcaggkgggg gtgccgggcg gggcggggcc gcctcgggcc
     1261 ggggrgggct cgggggrggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
     1321 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
     1381 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
     1441 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
     1501 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
     1561 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
     1621 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
     1681 gttattgtgc tgtctcatca ttttggcaaa gaattgagct cgatatcggt acgccaccat
     1741 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1801 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1861 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1921 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1981 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     2041 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2101 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2161 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2221 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2281 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2341 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2401 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gatgagctgt acaagggaag
     2461 cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc
     2521 tatgaaatct gcagagtatc tgaatacgtt ccgccttagg aatttgggcc tccccgtgat
     2581 gaacaatctc cacgatatga gcaaggcgac tcgaatatcc gtggaaacgc tgagactgct
     2641 catctataca gcagactttc ggtacaggat ctacacggtc gaaaagaagg ggcctgagaa
     2701 acgcatgcga acaatttatc aacctagccg agagctcaag gcgttgcagg gctgggttct
     2761 tcgaaacatc cttgacaaac tctcatcatc accctttagt attgggtttg aaaagcacca
     2821 aagcatcctt aacaacgcga cgccacacat aggtgccaat ttcatattga acatcgactt
     2881 ggaggatttt tttccgagcc tcacagccaa taaagtgttc ggtgtttttc acagtcttgg
     2941 gtacaatcgc cttattagtt ccgttcttac caagatttgt tgttacaaga atctcttgcc
     3001 ccagggagca cccagcagtc cgaaattggc gaatttgatt tgttccaagc tcgattatcg
     3061 aatacaaggg tacgcgggca gccggggact catctatacc cgctacgcag acgatcttac
     3121 gctgtctgcc caatcaatga agaaggtcgt aaaggcgcgg gatttcttgt tttctatcat
     3181 cccgtccgag ggcttggtaa ttaattccaa aaagacttgt atctcaggac cacgatctca
     3241 gcgaaaagtg acaggactcg tcatttctca agaaaaagtc ggtataggga gagagaagta
     3301 taaggaaatc cgcgcgaaga tccaccacat attctgtggc aagagcagcg agatagaaca
     3361 cgtccgaggc tggttgtcct tcatactgag cgtggactca aaaagccacc gccggttgat
     3421 cacctatatt tcaaaactgg aaaagaaata tggaaagaac ccactcaaca aagctaaaac
     3481 accaccaaag aagaaaagaa aggtctgatc ccggttagta atgagtttaa acctgtgcct
     3541 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3601 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3661 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3721 aatagcaggc atgctgggga tgcggtgggc tctatggaga tctgcgaaat cggatccagc
     3781 tgaatctaag tgaacgctga cgtcatcaac ccgctccaag gaatcgcggg cccagtgtca
     3841 ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa gatggctgtg agggacaggg
     3901 gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct gggaaatcac cataaacgtg
     3961 aaatgtcttt ggatttggga atcttataag ttctgtatga gaccacgaaa tgataagatt
     4021 ccgtatgcgc acccttagcg agaggtttat cattaaggtc aacctctgga tgttgtttcg
     4081 gcatcctgca ttgaatctga gttactgtct gttttcctac acacctcgtg actgtctacg
     4141 ccacgtagga cacatggtta tctggggtaa aggcagtaca tcatccctcc attgttcact
     4201 ctgggggttc atcacctcaa ccattatgtc aggaaacccg tttcttctga cgtaagggtg
     4261 cgcatacgga atcttatcat ctggggtaaa ggctgtagtt tcagagctat gctggaaaca
     4321 gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     4381 tgcttttttt tttttagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4441 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4501 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4561 ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc
     4621 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4681 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4741 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc
     4801 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4861 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     4921 ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4981 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5041 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5101 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5161 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5221 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5281 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5341 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5401 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5461 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5521 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5581 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5641 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5701 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5761 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5821 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5881 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5941 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6001 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6061 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6121 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6181 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6241 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6301 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6361 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6421 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6481 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6541 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6601 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6661 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6721 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6781 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6841 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6901 gtgccacctg ggtcgacatt gattattg
//
